Real-Time PCR Systems

Applied Biosystems 7900HT Fast Real-Time PCR System

and 7300/7500 Real-Time PCR Systems

Chemistry Guide
© Copyright 2005 Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This
document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or
consequential damages in connection with or arising from the use of this document.

NOTICE TO PURCHASER:
PLEASE REFER TO THE APPLIED BIOSYSTEMS 7900HT FAST REAL-TIME PCR SYSTEM AND SDS ENTERPRISE DATABASE USER GUIDE, OR
THE APPLIED BIOSYSTEMS 7300/7500/7500 FAST REAL-TIME PCR SYSTEM GETTING STARTED GUIDES FOR LIMITED LABEL LICENSE OR
DISCLAIMER INFORMATION.
The PCR process and 5′ nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-LaRoche Ltd.

TRADEMARKS:
ABI PRISM, Applied Biosystems, Celera, Primer Express, MicroAmp, and VIC are registered trademarks and Celera Discovery System, FAM, JOE, MultiScribe, NED,
ROX, TAMRA, and TET are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries.
AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.
SYBR and Texas Red are registered trademarks of Molecular Probes, Inc.
All other trademarks are the sole property of their respective owners.

Part Number 4348358 Rev. E

5/2005

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Contents

Preface
How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Safety and EMC Compliance Information

Safety Conventions Used in This Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
General Instrument Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
Biological Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

Chapter 1 Introduction
Selecting an Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
Selecting the Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Selecting an Assay Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Performing the Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9
Selecting a Data Analysis Approach and Determining Results . . . . . . . . . . . . . . . . . . . 1-9

Chapter 2 Chemistry Overview

SYBR Green I Dye Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
TaqMan Probe-Based Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
Selecting the Appropriate Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5
Minimizing DNA Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

Chapter 3 Gene Expression and Other Quantitative Assays

Section 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
About Quantification Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
Selecting a Quantification Assay Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6
Purchasing a Preformulated or Custom-Designed Quantification Assay . . . . . . . . . . . 3-7
TaqMan Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7

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 Custom TaqMan Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
Designing Your Own Quantification Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10
Primer and Probe Design Using Primer Express Software . . . . . . . . . . . . . . . . . . 3-11
Selecting the Appropriate Reagent Configuration . . . . . . . . . . . . . . . . . . . . . . . . 3-14
Using the Universal Thermal Cycling Parameters . . . . . . . . . . . . . . . . . . . . . . . . 3-16
Optimizing Primer Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21
Optimizing the Probe Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24
Using Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-27

Section 3.2 Selecting a Data Analysis Approach and Determining Results . . . . . . 3-31
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-32
Relative or Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-32
Relative Standard Curve Method for Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . 3-36
Comparative CT Method for Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . 3-42
Multiplex PCR (Same-Tube) Method for Relative Quantification . . . . . . . . . . . . . . . . 3-47
Standard Curve Method for Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . 3-53

Chapter 4 Allelic Discrimination Assays

About Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
Purchasing an Applied Biosystems TaqMan SNP Genotyping Assay Product . . . . . . . 4-4
TaqMan SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4
Custom TaqMan SNP Genotyping Assays Service . . . . . . . . . . . . . . . . . . . . . . . . 4-6
TaqMan Pre-Developed Assay Reagents for Allelic Discrimination . . . . . . . . . . . . . . . 4-6
Designing Your Own Allelic Discrimination Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6
Probe Design Using Primer Express Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7
Primer Design Using Primer Express Software . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8
Selecting the Appropriate Reagent Configuration . . . . . . . . . . . . . . . . . . . . . . . . . 4-9
Using the Universal Thermal Cycling Parameters . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Chapter 5 Plus/Minus Assays

About Plus/Minus Assays Using an IPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
Purchasing the Applied Biosystems TaqMan Exogenous IPC Reagents Kit . . . . . . . . 5-4

Chapter 6 Troubleshooting
Troubleshooting Quantification Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2
Troubleshooting Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7

Appendix A Formulas
Comparative CT Method for Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . A-1

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Appendix B Part Numbers
Real-Time PCR Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2
Sequence Detection System Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-3
Real-Time PCR Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-4
Real-Time PCR Reagent Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-5
Real-Time PCR RT-PCR Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-7
Real-Time PCR Reaction Kits (with Controls) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-8
Real-Time PCR Control Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-9
Real-Time PCR Reagent Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-10
Real-Time PCR Calibration Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-11
Real-Time PCR Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-12
TaqMan Pre-Developed Assay Reagents for Allelic Discrimination . . . . . . . . . . . . . . B-13
Custom Oligonucleotide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-14

Appendix C References

Index

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Preface

How to Use This Guide

Purpose of This The Applied Biosystems Real-Time PCR Systems Chemistry Guide provides an easy-
Guide to-use reference on various techniques and applications, including:
• An introduction to real-time PCR chemistries
• Background information, design guidelines, and general procedures for the
following assay types:
– Gene Expression and Quantification Assays
– Allelic Discrimination Assays
– Plus/Minus Assays
• Troubleshooting information

Audience This guide is intended for users of Applied Biosystems Real-Time PCR Systems
instruments and chemistries who have a working knowledge of the polymerase chain
reaction (PCR) process.

Text Conventions This guide uses the following conventions:

• Bold indicates user action. For example:
Type 0, then press Enter for each of the remaining fields.
• Italic text indicates new or important words and is also used for emphasis. For
example:
Before analyzing, always prepare fresh matrix.
• A right arrow bracket (>) separates successive commands you select from a
drop-down or shortcut menu. For example:
Select File > Open > Spot Set.
Right-click the sample row, then select View Filter > View All Runs.

User Attention Two user attention words appear in Applied Biosystems user documentation. Each
Words word implies a particular level of observation or action as described below:
Note: Provides information that may be of interest or help but is not critical to the
use of the product.
IMPORTANT! Provides information that is necessary for proper instrument
operation, accurate chemistry kit use, or safe use of a chemical.

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Preface

Examples of the user attention words appear below:

Note: The size of the column affects the run time.
Note: The Calibrate function is also available in the Control Console.
IMPORTANT! To verify your client connection to the database, you need a valid
Oracle user ID and password.
IMPORTANT! You must create a separate Sample Entry Spreadsheet for each 96-well
microtiter plate.

Safety Alert Safety alert words also appear in user documentation. For more information, see
Words “Safety Alert Words” on page xii.

How to Obtain More Information

Related When using this chemistry guide, you may find the following documents to be
Documentation helpful references.
• Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise
Database User Guide (PN 4351684)
• Applied Biosystems 7900HT Fast Real-Time PCR System User Bulletin:
Performing Fast Gene Quantification (PN 4352533)
• RQ Manager Software User Guide (PN 4351670)
• SNP Manager Software User Guide (PN 4351671)
• Primer Express® Software Version 3.0 Getting Started Guide (PN 4362460)
• Applied Biosystems 7500 Fast Real-Time PCR System Performing Fast Gene
Quantification Quick Reference Card (PN 4362285)
• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative
Quantification Getting Started Guide (PN 4347824)
• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus
Getting Started Guide (PN 4347821)
• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Allelic
Discrimination Getting Started Guide (PN 4347822)
• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute
Quantification Getting Started Guide (PN 4347825)
• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation
and Maintenance Getting Started Guide (PN 4347828)
• TaqMan® Fast Universal PCR Master Mix (2✕) Protocol (PN 4351891)
• TaqMan® Gene Expression Assays Protocol (PN 4333458)
• TaqMan® SNP Genotyping Assays Protocol (PN 4332856)
Note: For additional documentation, see “How to Obtain Support” on page ix.

Send Us Your Applied Biosystems welcomes your comments and suggestions for improving its
Comments user documents. You can e-mail your comments to:
techpubs@appliedbiosystems.com

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 How to Obtain Support

How to Obtain Support

For the latest services and support information for all locations, go to
http://www.appliedbiosystems.com, then click the link for Support.
At the Support page, you can:
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order Applied Biosystems user documents, MSDSs, certificates of analysis,
and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
In addition, the Support page provides access to worldwide telephone and fax
numbers to contact Applied Biosystems Technical Support and Sales facilities.

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Preface

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Safety and EMC Compliance Information

This section includes the following topics:

Safety Conventions Used in This Document. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
General Instrument Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
Biological Hazard Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

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Safety and EMC Compliance Information

Safety Conventions Used in This Document

Safety Alert Four safety alert words appear in Applied Biosystems user documentation at points
Words in the document where you need to be aware of relevant hazards. Each alert
word–IMPORTANT, CAUTION, WARNING, DANGER–implies a particular
level of observation or action, as defined below:

Definitions
IMPORTANT! – Indicates information that is necessary for proper instrument
operation, accurate chemistry kit use, or safe use of a chemical.

– Indicates a potentially hazardous situation that, if not avoided,

may result in minor or moderate injury. It may also be used to alert against unsafe
practices.

– Indicates a potentially hazardous situation that, if not avoided,

could result in death or serious injury.

– Indicates an imminently hazardous situation that, if not avoided,

will result in death or serious injury. This signal word is to be limited to the most
extreme situations.
Except for IMPORTANTs, each safety alert word in an Applied Biosystems
document appears with an open triangle figure that contains a hazard symbol. These
hazard symbols are identical to the hazard icons that are affixed to Applied
Biosystems instruments.

Examples
The following examples show the use of safety alert words:
IMPORTANT! You must create a separate a Sample Entry Spreadsheet for each
96-well plate.

The lamp is extremely hot. Do not touch the lamp until it has
cooled to room temperature.

CHEMICAL HAZARD. Formamide. Exposure causes eye,

skin, and respiratory tract irritation. It is a possible developmental and birth defect
hazard. Read the MSDS, and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves.

ELECTRICAL HAZARD. Failure to ground the instrument

properly can lead to an electrical shock. Ground the instrument according to the
provided instructions.

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 General Instrument Safety

General Instrument Safety

PHYSICAL INJURY HAZARD. Use this product only as
specified in this document. Using this instrument in a manner not specified by
Applied Biosystems may result in personal injury or damage to the instrument.

Chemical Safety
Chemical Hazard CHEMICAL HAZARD. Before handling any chemicals, refer
Warning to the Material Safety Data Sheet (MSDS) provided by the manufacturer, and
observe all relevant precautions.

CHEMICAL HAZARD. All chemicals in the instrument,

including liquid in the lines, are potentially hazardous. Always determine what
chemicals have been used in the instrument before changing reagents or instrument
components. Wear appropriate eyewear, protective clothing, and gloves when
working on the instrument.

CHEMICAL HAZARD. Four-liter reagent and waste bottles

can crack and leak. Each 4-liter bottle should be secured in a low-density
polyethylene safety container with the cover fastened and the handles locked in the
upright position. Wear appropriate eyewear, clothing, and gloves when handling
reagent and waste bottles.

CHEMICAL STORAGE HAZARD. Never collect or store

waste in a glass container because of the risk of breaking or shattering. Reagent and
waste bottles can crack and leak. Each waste bottle should be secured in a low-
density polyethylene safety container with the cover fastened and the handles locked
in the upright position. Wear appropriate eyewear, clothing, and gloves when
handling reagent and waste bottles.

About MSDSs Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with
shipments of hazardous chemicals to new customers. They also provide MSDSs with
the first shipment of a hazardous chemical to a customer after an MSDS has been
updated. MSDSs provide the safety information you need to store, handle, transport,
and dispose of the chemicals safely.
Each time you receive a new MSDS packaged with a hazardous chemical, be sure to
replace the appropriate MSDS in your files.

Obtaining You can obtain from Applied Biosystems the MSDS for any chemical supplied by
MSDSs Applied Biosystems. This service is free and available 24 hours a day.
To obtain MSDSs:
1. Go to https://docs.appliedbiosystems.com/msdssearch.html
2. In the Search field, type in the chemical name, part number, or other
information that appears in the MSDS of interest. Select the language of your
choice, then click Search.

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Safety and EMC Compliance Information

3. Find the document of interest, right-click the document title, then select any of
the following:
• Open – To view the document
• Print Target – To print the document
• Save Target As – To download a PDF version of the document to a
destination that you choose
4. To have a copy of a document sent by fax or e-mail, select Fax or Email to the
left of the document title in the Search Results page, then click RETRIEVE
DOCUMENTS at the end of the document list.
5. After you enter the required information, click View/Deliver Selected
Documents Now.

Chemical Safety To minimize the hazards of chemicals:

Guidelines • Read and understand the Material Safety Data Sheets (MSDS) provided by the
chemical manufacturer before you store, handle, or work with any chemicals or
hazardous materials. (See “About MSDSs” on page xiii.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood). For additional
safety guidelines, consult the MSDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer’s cleanup procedures as recommended on the MSDS.
• Comply with all local, state/provincial, or national laws and regulations related
to chemical storage, handling, and disposal.

Chemical Waste Safety

Chemical Waste HAZARDOUS WASTE. Refer to Material Safety Data Sheets
Hazard and local regulations for handling and disposal.

CHEMICAL WASTE HAZARD. Wastes produced by Applied

Biosystems instruments are potentially hazardous and can cause injury, illness, or
death.

CHEMICAL STORAGE HAZARD. Never collect or store

waste in a glass container because of the risk of breaking or shattering. Reagent and
waste bottles can crack and leak. Each waste bottle should be secured in a low-
density polyethylene safety container with the cover fastened and the handles locked
in the upright position. Wear appropriate eyewear, clothing, and gloves when
handling reagent and waste bottles.

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 Chemical Waste Safety

Chemical Waste To minimize the hazards of chemical waste:

Safety Guidelines • Read and understand the Material Safety Data Sheets (MSDSs) provided by the
manufacturers of the chemicals in the waste container before you store, handle,
or dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary waste container
holds the immediate waste. A secondary container contains spills or leaks from
the primary container. Both containers must be compatible with the waste
material and meet federal, state, and local requirements for container storage.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood).For additional
safety guidelines, consult the MSDS.
• Handle chemical wastes in a fume hood.
• After emptying the waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in accordance with
good laboratory practices and local, state/provincial, or national environmental
and health regulations.

Waste Disposal If potentially hazardous waste is generated when you operate the instrument, you
must:
• Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and disposed
of according to all local, state/provincial, and/or national regulations.
IMPORTANT! Radioactive or biohazardous materials may require special handling,
and disposal limitations may apply.
• Be aware that high solvent flow rates (~40 mL/min) may cause a static charge to
build up on the surface of the tubing. Electrical sparks may result.

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Safety and EMC Compliance Information

Biological Hazard Safety

General BIOHAZARD. Biological samples such as tissues, body fluids,
Biohazard and blood of humans and other animals have the potential to transmit infectious
diseases. Follow all applicable local, state/provincial, and/or national regulations.
Wear appropriate protective eyewear, clothing, and gloves. Read and follow the
guidelines in these publications:
• U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories (stock no. 017-040-
00547-4; http://bmbl.od.nih.gov)
• Occupational Safety and Health Standards, Bloodborne Pathogens
(29 CFR§1910.1030; http://www.access.gpo.gov/nara/cfr/
waisidx_01/29cfr1910a_01.html).

Additional information about biohazard guidelines is available at:

http://www.cdc.gov

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Introduction 1 1
This chapter covers:
Selecting an Assay Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2
Selecting the Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Selecting an Assay Source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Performing the Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9
Selecting a Data Analysis Approach and Determining Results . . . . . . . . . . . . . . . 1-9

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Chapter 1 Introduction

Selecting an Assay Type

Real-Time PCR Applied Biosystems has five instruments in its Real-Time PCR Systems product
Instruments line:
• Applied Biosystems 7900HT Fast Real-Time PCR System (7900HT System)
• ABI PRISM® 7900HT Sequence Detection System, upgradeable to the Applied
Biosystems 7900HT Fast Real-Time PCR System with the 7900HT System Fast
Service Upgrade
• Applied Biosystems 7300 Real-Time PCR System (7300 System)
• Applied Biosystems 7500 Real-Time PCR System (7500 System), upgradeable
to the Applied Biosystems 7500 Fast Real-Time PCR System with the 7500 Fast
Real-Time PCR Upgrade Kit
• Applied Biosystems 7500 Fast Real-Time PCR System (7500 Fast System)
Note: For details on these instruments, refer to the instrument user guides. See “How
to Obtain More Information” on page viii for a list of the user guide titles and part
numbers.

Assay Types The three assay types (quantification, allelic discrimination, and plus/minus) can be
Supported categorized into real-time PCR and endpoint assays as shown in Table 1-1.

Table 1-1 Assay types

Assay Category Function Procedure Description

Real-Time PCR Quantification, including: “About Quantification

Assays” on page 3-4
• One-step reverse
transcription polymerase
chain reaction
(RT-PCR) or RNA
quantification
• Two-step RT-PCR for
RNA quantification
• DNA quantification

Endpoint Assay Allelic Discrimination “About Allelic

Discrimination Assays” on
page 4-2

Plus/Minus “About Plus/Minus Assays

Using an IPC” on page 5-2

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 Selecting an Assay Type

Real-time PCR systems can be used to perform the assay types in Table 1-2.

Table 1-2 Real-time PCR systems and assay types

Real-Time Assay Endpoint Assay

Instrument
Allelic Plus/Minus
Gene Expression
Discrimination with IPC

7900HT System with Yes Yes Yes

Standard 96- or 384-Well
Block Module

7900HT System with Fast Yes Yes No

96-Well Block Module

7300 System Yes Yes Yes

7500 System Yes Yes Yes

7500 Fast System Yes Yes Yes

Table 1-3 Consumable types supported

Consumable

Optical 96-
Instrument 96-Well 384-Well TaqMan®
Well Fast Individual
Optical Optical Low
Thermal Optical
Reaction Reaction Density
Cycling Tubes
Plate Plate Array a
Plate

7900HT System No No Yes No No

with Standard 384-
Well Block

7900HT System Yes No No No No

with Standard 96-
Well Block

7900HT System No Yes No No No

with Fast 96-Well
Block

7900HT System No No No Yes No

with 7900HT
TaqMan® Low
Density Array Block

7300 System Yes No No No Yes

7500 System Yes No No No Yes

7500 Fast System No Yes No No No

a. For more information on the TaqMan Low Density Array, see the Applied Biosystems
7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide.

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Chapter 1 Introduction

About Real-Time Real-time PCR is the ability to monitor the progress of the PCR as it occurs. Data is
PCR Assays collected throughout the PCR process rather than at the end of the PCR process.
In real-time PCR, reactions are characterized by the point in time during cycling
when amplification of a target is first detected rather than the amount of target
accumulated after a fixed number of cycles.

About One-Step RT-PCR

RT-PCR is used to quantify RNA. RT-PCR can be performed as a one-step or two-
step procedure.
The one-step RT-PCR performs RT as well as PCR in a single buffer system
(Figure 1-1). The reaction proceeds without the addition of reagents between the RT
and PCR steps. One-step RT-PCR offers the convenience of a single-tube preparation
for RT and PCR amplification. However, the carryover prevention enzyme,
AmpErase® UNG (uracil-N-glycosylase), cannot be used with one-step RT-PCR. In
one-step RT-PCR, the presence of UNG would destroy the cDNA as it is being made.
For information about UNG, see “Using UNG to Minimize Reamplification
Carryover Products” on page 2-6.

Single
Tube

Figure 1-1 Schematic representation of one-step RT-PCR

About Two-Step RT-PCR

Two-step RT-PCR is performed in two separate reactions (Figure 1-2). Two-step RT-
PCR is useful when detecting multiple transcripts from a single cDNA reaction, or
when storing a portion of the cDNA for later use. When you perform PCR using
dUTP as a base in the RT step, you can use AmpErase UNG enzyme to prevent
carryover contamination. For information about UNG, see “Using UNG to Minimize
Reamplification Carryover Products” on page 2-6.

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 Selecting an Assay Type

Tube 1
Oligo d(T) or random hexamer

Tube 2 PCR Step

Figure 1-2 Schematic representation of two-step RT-PCR

Table 1-4 summarizes the differences between one- and two-step RT-PCR.

Comparison of RT-PCR Methods

Table 1-4 Primers for one- and two-step RT-PCR

Method Primers for cDNA Synthesis Comments

One-step Sequence-specific reverse primer Requires single reaction mix

UNG cannot be used

Two-step Random hexamers cDNA can be stored for later use

UNG can be used
Oligo d(T)16
Requires two reaction mixes
Sequence-specific reverse primers

About An endpoint assay (also called a plate read assay) measures the amount of
Endpoint Assays accumulated PCR product in fluorescence units at the end of the PCR process. The
datapoint is the normalized intensity of the reporter dye, or Rn.
Some endpoint assays can include both pre-PCR and post-PCR datapoints. In this
case, the system calculates the delta Rn (∆Rn) value per the following formula:
Rn post-PCR – Rn pre-PCR = ∆Rn

About Multiplex Multiplex PCR is the use of more than one primer/probe set in the same tube.
PCR Multiplex PCR is most commonly used in 5′ nuclease quantification assays that
involve relative quantification of gene expression.
Typically one probe is used to detect the target species; another probe is used to
detect an endogenous control (internal control gene). Running both assays in a single
tube reduces both the running costs and the dependence on accurate pipetting when
splitting a sample into two separate tubes.

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Chapter 1 Introduction

Selecting the Chemistry

Applied Biosystems has developed two types of chemistries that can be used to
detect PCR products on real-time PCR instruments:
• TaqMan Probe-based fluorogenic 5′ nuclease chemistry
• SYBR® Green I dye chemistry
These chemistries are discussed in detail in Chapter 2.

TaqMan Probe- Applied Biosystems TaqMan Probe-based chemistry uses a fluorogenic probe to
Based Chemistry enable the detection of a specific PCR product as it accumulates during PCR cycles.
The Applied Biosystems patented fluorogenic probe design, which incorporates the
reporter dye on the 5′ end and the quencher dye on the 3′ end, has greatly simplified
the design and synthesis of effective 5′ fluorogenic nuclease assay probes (Livak,
Flood, et al., 1995).

Assay Types that Use TaqMan Probe-Based Chemistry

The TaqMan Probe-based chemistry can be used for the following assay types:
• Quantification, including:
– One-step RT-PCR for RNA quantification
– Two-step RT-PCR for RNA quantification
– DNA quantification
• Allelic Discrimination
• Plus/Minus

SYBR Green I The SYBR Green I dye chemistry uses SYBR Green I dye, which binds to double-
Dye Chemistry stranded DNA, to detect PCR products as they accumulate during PCR cycles.
An important difference between the TaqMan probes and SYBR Green I dye
chemistries is that the SYBR Green I dye chemistry binds all double-stranded DNA,
including nonspecific reaction products. A well-optimized reaction is therefore
essential for accurate results.You can not perform multiplex PCR using SYBR Green
I dye.

Assay Types that Use SYBR Green I Dye Chemistry

The SYBR Green I dye chemistry can be used for quantification assay types
including:
• One-step RT-PCR for RNA quantification
• Two-step RT-PCR for RNA quantification
• DNA quantification

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 Selecting an Assay Source

Selecting an Assay Source

After you select an assay type and chemistry, you can:
• Purchase predesigned probes and primers (TaqMan® Gene Expression Assays
and TaqMan® SNP Genotyping Assays)
• Use the Applied Biosystems Custom TaqMan® Gene Expression Assays or
Custom TaqMan® SNP Genotyping Assays service to custom-design probes and
primers, based on your sequence submission.
• Design your own TaqMan probe and primers using Primer Express® software,
then purchase the probe and primers.
Note: TaqMan Gene Expression and Custom TaqMan Gene Expression Assays are
intended for use in singleplex reactions.

TaqMan Gene TaqMan Gene Expression and TaqMan SNP Genotyping Assays provide the most
Expression and comprehensive collection of biologically informative, predesigned, quality-
TaqMan SNP controlled, and validated probes and primers ready to use on an Applied Biosystems
Genotyping Real-Time PCR System.
Assays All assays are designed using Applied Biosystems powerful bioinformatics pipeline
and software, incorporating information from the Celera Discovery System™ Online
Platform (CDS) and public databases.
TaqMan® Assays include:
• TaqMan SNP Genotyping Assays (PN 4331183 and PN 4351379) for
genotyping single nucleotide polymorphisms (SNPs). The products use the
5′ nuclease assay for amplifying and detecting specific SNP alleles in purified
human genomic DNA samples. Each assay allows researchers to genotype
individuals for a specific SNP.
Each primer and probe set was validated on two to four populations, each
consisting of approximately 45 genomic DNA samples to ensure the highest
quality and to provide population-specific allele frequency information.
• TaqMan Gene Expression Assays (PN 4331182 and 4351372) are a
comprehensive collection of predesigned gene-specific primer and probe sets
for quantitative gene expression studies on human, mouse, rat, Arabidopsis, and
Drosophila genes. TaqMan Gene Expression Assays are built on Applied
Biosystems 5′ nuclease chemistry. Each assay consists of two unlabeled PCR
primers and one FAM™ dye-labeled probe. All components are quality-control
tested and formulated as a 20✕ mix. Additionally, a number of
TaqMan® Endogenous Controls are available for all species, with either
VIC® dye-labeled or FAM™ dye-labeled TaqMan® MGB (minor groove binder)
probes. TaqMan Endogenous Controls with VIC dye labels are primer-limited.
For information on available products and specific product uses, contact your
Applied Biosystems representative or visit the Applied Biosystems web site. See
“How to Obtain Support” on page ix.

Custom TaqMan Custom TaqMan SNP Genotyping and Custom TaqMan Gene Expression Assays are
SNP Genotyping assay development services that design, synthesize, formulate and deliver
and Gene analytically-controlled primer and probe sets for SNP genotyping and gene
Expression expression assays based on sequence information submitted by the customer. For
Assays human SNP genotyping assays, an additional functional test is performed.

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Chapter 1 Introduction

• Customers submit target sequences using File Builder, a free application

available on the Applied Biosystems Web site (available for download from the
Custom TaqMan Genotyping and Gene Expression Assays Web pages).
• Customers can submit any number of target sequences, either directly to the
eStore (through File Builder) or by e-mailing or regularly mailing the File
Builder text file to the regional Applied Biosystems sales office. Ordering
details are in the File Builder Web Help Guide or in the Ordering Information
sidebar link on the Custom TaqMan Genotyping and Gene Expression Assays
Web pages.
• Assays are available in three scales: small (20✕, 750 µL), medium (40✕, 3 mL),
and large (80✕, 12 mL).
• Applied Biosystems provides ready-to-use probe and primer sets for each target
sequence in a single tube, along with an assay information file, which contains
information about each assay in the order, including primer and probe
sequences.
• Customers are charged only for probe and primer sets that successfully pass
manufacturing quality controls.
Visit the following Applied Biosystems Web sites for more information about
Custom TaqMan SNP Genotyping Assays:
http://www.allsnps.com
and Custom TaqMan Gene Expression Assays:
http://www.allgenes.com

Designing Your When designing your own assay, follow the assay design guidelines described in this
Own Assay document. These guidelines have been developed by Applied Biosystems to optimize
results when using real-time PCR instruments and TaqMan probes or SYBR Green I
dye chemistries.
Applied Biosystems assay design guidelines do not guarantee that all assays will
provide the same level of performance and sensitivity. Even the most scrupulous
design parameters cannot account for all the possible variables that can exist between
two different assay systems.
Guidelines for quantification and allelic discrimination are described in “Designing
Your Own Quantification Assay” on page 3-10 and “Designing Your Own Allelic
Discrimination Assay” on page 4-6.

Important Design Steps

Applied Biosystems Assay Design Guidelines specify that you:
• Design primers and probes using Primer Express® Software – The Primer
Express software is used to design primers and probes. The software uses a set
of default parameters to automatically select primer and probe sets.
• Select the appropriate reagent configuration – There are several TaqMan and
SYBR Green I dye chemistry kits available. The reagent configuration you use
depends on your assay type. For quantification assays, see “Selecting the
Appropriate Reagent Configuration” on page 3-14.

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 Performing the Assay

• Use universal thermal cycling parameters – All assay types designed using
Applied Biosystems assay design guidelines can be run using universal thermal
cycling parameters. This protocol design eliminates the need to optimize the
thermal cycling parameters and allows multiple assays to be run on the same
plate without sacrificing performance. Fast PCR thermal cycling parameters are
unique to Fast applications.
• Use default primer and probe concentrations or optimize primer and probe
concentrations – When you use Applied Biosystems assay design guidelines,
you can use default primer and probe concentrations for non-multiplex
optimized assays, or you can optimize primer and probe concentrations. For
quantification assays, see “Optimizing Primer Concentrations” on page 3-21
and “Optimizing the Probe Concentration” on page 3-24.
IMPORTANT! To achieve the highest level of success, use all the assay design
guidelines together because many of the individual components of the system are
interdependent.
To illustrate this point, consider the following example. The ability to use universal
thermal cycling parameters is based on the assumption that the selected primers have
a melting temperature (Tm) of 58 to 60 °C as calculated by Primer Express software.
If the primers do not have the correct Tms, or even if the Tms have been calculated
with a primer design software package other than Primer Express, optimal
performance and even functionality of the assay cannot be assured.
Visit the Applied Biosystems support web site to access a variety of tutorials on how
to use the Primer Express software for designing real-time quantitative assays. See
“How to Obtain Support” on page ix.

Performing the Assay

For information about performing the assay on your system, see the documentation
provided with your system. For information about laboratory practices, see
“Minimizing DNA Contaminants” on page 2-6.

Selecting a Data Analysis Approach and Determining

Results
For information, see Section 3.2, “Selecting a Data Analysis Approach and
Determining Results,” on page 3-31.

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Chapter 1 Introduction

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Chemistry Overview 2 2
This chapter covers:
SYBR Green I Dye Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2
TaqMan Probe-Based Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
Selecting the Appropriate Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5
Minimizing DNA Contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6

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Chapter 2 Chemistry Overview

SYBR Green I Dye Chemistry

Development of Small molecules that bind to double-stranded DNA can be divided into two classes:
SYBR Green I intercalators and minor groove-binders (MGBs). Higuchi (Higuchi et al., 1992) used
Dye Chemistry the intercalator ethidium bromide for their real-time detection of PCR. Hoechst
33258 is an example of a minor groove-binding dye whose fluorescence increases
when bound to double-stranded DNA (Higuchi et al., 1993).
Regardless of the binding method, there are at least two requirements for a DNA
binding dye for real-time detection of PCR products:
• Increased fluorescence when bound to double-stranded DNA
• No inhibition of PCR
Applied Biosystems has developed conditions that permit the use of the SYBR®
Green I dye in PCR without PCR inhibition and with increased sensitivity of
detection compared with ethidium bromide.

How the SYBR The SYBR Green I dye chemistry uses the SYBR Green I dye to detect PCR
Green I Dye products by binding to double-stranded DNA formed during PCR. Here’s how it
Chemistry Works works:
1. When SYBR Green PCR Master Mix is added to a sample, SYBR Green I dye
immediately binds to all double-stranded DNA.
2. During the PCR, AmpliTaq Gold® DNA Polymerase amplifies the target
sequence, which creates the PCR product, or “amplicon.”
3. The SYBR Green I dye then binds to each new copy of double-stranded DNA.
4. As the PCR progresses, more amplicon is created.
Since the SYBR Green I dye binds to all double-stranded DNA, the result is an
increase in fluorescence intensity proportional to the amount of double-stranded
PCR product produced.
Figure 2-1 illustrates this process.

Step 1 Step 2 Step 3

The SYBR Green I dye within the During PCR, AmpliTaq Gold The SYBR Green I dye then
SYBR Green PCR Master Mix DNA Polymerase amplifies binds to each new copy of
immediately binds with all each target. double-stranded DNA.
double-stranded DNA present in
the sample.

Figure 2-1 Representation of how the SYBR Green I dye acts on double-
stranded DNA during one extension phase of PCR

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 TaqMan Probe-Based Chemistry

TaqMan Probe-Based Chemistry

Development of Initially, intercalator dyes were used to measure real-time PCR products. The
TaqMan Probe- primary disadvantage to these type of probes is that they detect accumulation of both
Based Chemistry specific and nonspecific PCR products.
Real-time systems for PCR were improved by the introduction of fluorogenic-labeled
probes that use the 5′ nuclease activity of the Applied Biosystems hot-start DNA
polymerase system. The availability of these fluorogenic probes enabled the
development of a real-time method for detecting only specific amplification
products.

How TaqMan The TaqMan® Probe-based chemistry uses a fluorogenic probe to enable the
Real-Time detection of a specific PCR product as it accumulates during PCR. Here is how it
Chemistry Works works (Figure 2-2):
1. An oligonucleotide probe is constructed with a fluorescent reporter dye bound
to the 5′ end and a quencher on the 3′ end.
While the probe is intact, the proximity of the quencher greatly reduces the
fluorescence emitted by the reporter dye by fluorescence resonance energy
transfer (FRET; Förster resonance, Förster, V. T. 1948) through space.
2. If the target sequence is present, the probe anneals between primer sites and is
cleaved by the 5′ nuclease activity of Applied Biosystems hot-start DNA
polymerase system during extension.
3. This cleavage of the probe:
– Separates the reporter dye from the quencher, increasing the reporter dye
signal.
– Removes the probe from the target strand, allowing primer extension to
continue to the end of the template strand. Thus, inclusion of the probe does
not inhibit the overall PCR process.
4. Additional reporter dye molecules are cleaved from their respective probes with
each cycle, resulting in an increase in fluorescence intensity proportional to the
amount of amplicon produced. The higher the starting copy number of the
nucleic acid target, the sooner a significant increase in fluorescence is observed.
Figure 2-2 illustrates this process.

POLYMERIZATION STRAND DISPLACEMENT CLEAVAGE POLYMERIZATION COMPLETED

R R Q
FORWARD R = REPORTER R 3'
PRIMER R PROBE Q Q = QUENCHER Q Q
5' 3' 5' 3' 5' 3' 5'
3' 5' 3' 5' 3' 5' 3' 5'

5' 3' 5' 3' 5' 3' 5' 3'

5' 5' 5' 5'
REVERSE PRIMER

Step 1: A reporter (R) and a Step 2: When both dyes are Step 3: During each extension Step 4: Once separated from the
quencher (Q) are attached to the
5′ and 3′ ends of a TaqMan
attached to the probe, reporter dye cycle, the Applied Biosystems quencher, the reporter dye emits
emission is quenched. hot-start DNA polymerase system its characteristic fluorescence.
probe. cleaves the reporter dye from the
probe.

Figure 2-2 Representation of how the 5′ nuclease chemistry uses a fluorogenic

probe to enable detection of a specific PCR product

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Chapter 2 Chemistry Overview

Two Types of Applied Biosystems offers two types of TaqMan probes (Table 2-1):
TaqMan Probes • TaqMan probes with TAMRA™ dye as quencher
• TaqMan MGB (minor groove-binder) probes with non fluorescent quencher
(NFQ)

Table 2-1 Types of TaqMan probes

5′ Label Dye 3′ Label Dye Other Features

Custom Custom FAM™, TET™ TAMRA —

Probes TaqMan® Probe or VIC®

Custom FAM™, TET™, Nonfluorescent minor groove-

TaqMan® MGB NED™, or VIC® quencher binder
Probe

TaqMan Gene FAM Nonfluorescent minor groove-

Assays Expression quencher binder
(TaqMan MGB)
SNP FAM, VIC Nonfluorescent minor groove-
Genotyping quencher binder

Custom Gene FAM Nonfluorescent minor groove-

TaqMan Expression quencher binder
Assays
(TaqMan MGB) SNP FAM, VIC Nonfluorescent minor groove-
Genotyping quencher binder

TaqMan MGB Probes Recommended

Applied Biosystems recommends the general use of TaqMan MGB probes,
especially when conventional TaqMan probes exceed 30 nucleotides. The TaqMan
MGB probes contain:
• A nonfluorescent quencher at the 3′ end
Allows the Real-Time PCR instruments to measure the reporter dye
contributions more precisely because the quencher does not fluoresce.
• A minor groove-binder at the 3′ end
Increases the melting temperature (Tm) of probes (Afonina et al., 1997;
Kutyavin et al., 1997), allowing the use of shorter probes. Consequently, the
TaqMan MGB probes exhibit greater differences in Tm values between matched
and mismatched probes, which provides more accurate allelic discrimination.

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 Selecting the Appropriate Chemistry

Selecting the Appropriate Chemistry

The SYBR Green I dye and TaqMan Probe-based chemistry can be used for the assay
types listed in Table 2-2. See Table 2-3 for considerations for choosing SYBR Green
or TaqMan Probe-based chemistry.

Table 2-2 Assay type using SYBR Green 1 dye or TaqMan Probe-based
chemistry

Assay Type

Allelic Plus/Minus
Quantification a
Chemistry Discrimination using an IPC
(Chapter 3)
(Chapter 4) (Chapter 5)

SYBR Green I Dye Yes No No

TaqMan probes Yes Yes Yes

a. Includes one-step reverse transcription polymerase chain reaction (RT-PCR) and two-step
RT-PCR for RNA quantification and DNA/cDNA quantification

Table 2-3 Chemistry considerations for quantification assays using SYBR

Green 1 dye or TaqMan Probe-based chemistry

Chemistry Advantage Limitation

SYBR Green I • Provides amplification of any • Can bind to nonspecific

double-stranded DNA sequence double-stranded DNA
• Dissociation curves yield melting sequences. To avoid false
profile of distinct PCR products; positive signals, check for
allows melt curves to be added on nonspecific product
the run formation using
dissociation curve or gel
• Increases sensitivity for detecting analysis
amplification products relative to
product length • Multiplex reactions are not
supported
• Provides a convenient and cost
effective real-time chemistry to • Optimized assays are not
screen many samples and targets available

TaqMan • Increases specificity with a probe • Requires synthesis of a

probe • Provides multiplex capability; allows unique probe
for labeling probes with different,
distinguishable reporter dyes, which
allows detection of two distinct
sequences in one reaction tube
• Optimized assays available
• Allows 5′ nuclease assay to be
carried out during PCR

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Chapter 2 Chemistry Overview

Minimizing DNA Contaminants

The DNA amplification capability of the PCR process makes special laboratory
practices necessary when performing assays using fluorogenic 5′ nuclease (TaqMan
Probe-based chemistry) or SYBR Green I dye chemistry. Potential contamination can
be introduced by samples with high DNA concentrations, either from the DNA
template controls or from PCR carryover.
In addition, due to the nonspecific nature of the SYBR Green I dye, any double-
stranded DNA will be detected. When using the SYBR Green I dye chemistry, check
for nonspecific product formation by using dissociation curve or gel analysis. Care
must be taken to avoid contamination with target DNA. Gene expression assays that
span exon-exon junctions minimize the effect of gDNA (genomic DNA)
contaminants.

Using UNG to AmpErase® uracil-N-glycosylase (UNG) is a 26-kDa recombinant enzyme encoded

Minimize by the Escherichia coli uracil-N-glycosylase gene. This gene has been inserted into
Reamplification an E. coli host to direct expression of the native form of the enzyme (Kwok and
Carryover Higuchi, 1989).
Products UNG acts on single- and double-stranded dU-containing DNA. It acts by hydrolyzing
uracil-glycosidic bonds at dU-containing DNA sites. The enzyme causes the release
of uracil, thereby creating an alkali-sensitive apyrimidic site in the DNA. The
enzyme has no activity on RNA or dT-containing DNA (Longo et al., 1990).

TaqMan® Assays
For 5′ nuclease assays (which use either the TaqMan® 2✕ Universal PCR Master Mix
or the TaqMan® Fast Universal PCR Master Mix (2✕), No AmpErase® UNG),
AmpErase UNG treatment can prevent the re-amplification of carryover PCR
products. When dUTP replaces dTTP in PCR amplification, AmpErase UNG
treatment can remove up to 200,000 copies of amplicon per 50-µL reaction.
Note: TaqMan 2✕ Universal PCR Master Mix is available with or without
AmpErase UNG. If you are using TaqMan Fast Universal PCR Master Mix (2✕), No
AmpErase UNG, you must purchase AmpErase UNG separately.

SYBR Green I Dye Assays

AmpErase UNG treatment can also be useful in preventing the re-amplification of
carryover PCR products in SYBR Green I dye assays. Although the SYBR Green
PCR Master Mix does not contain AmpErase UNG, dTTP has been replaced with
dUTP, thus making the SYBR Green PCR Master Mix compatible with the use of
AmpErase UNG. If contamination from PCR carryover is suspected, use
AmpErase UNG to troubleshoot the problem.
Note: AmpErase UNG can be purchased individually (PN N808-0096) or as part of
the SYBR Green PCR Core Reagents kit (PN 4304886).

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 Minimizing DNA Contaminants

General PCR Use the following precautions to minimize sample contamination and PCR product
Practices carryover:
• Wear a clean lab coat (not previously worn while handling amplified PCR
products or used during sample preparation) and clean gloves when preparing
samples for PCR amplification. Change gloves whenever you suspect that they
are contaminated.
• Maintain separate areas, dedicated equipment, and supplies for:
– Sample preparation.
– PCR setup. Never bring amplified PCR products into the PCR setup area.
– PCR amplification.
– Analysis of PCR products.
• Open and close all sample tubes carefully. Avoid splashing or spraying PCR
samples.
• Use positive-displacement or air-displacement pipettors with filter-plugged tips.
Change tips after each use.
• Keep reactions and components capped as much as possible.
• Clean lab benches and equipment periodically with 10% bleach solution or 70%
ethanol.

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Chapter 2 Chemistry Overview

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Gene Expression and Other
Quantitative Assays 3 3
Section 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
About Quantification Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
Selecting a Quantification Assay Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6
Purchasing a Preformulated or Custom-Designed Quantification Assay . . . . . . . . 3-7
TaqMan Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7
Custom TaqMan Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
Designing Your Own Quantification Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10
Primer and Probe Design Using Primer Express Software . . . . . . . . . . . . . 3-11
Selecting the Appropriate Reagent Configuration . . . . . . . . . . . . . . . . . . . 3-14
Using the Universal Thermal Cycling Parameters . . . . . . . . . . . . . . . . . . . 3-16
Optimizing Primer Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21
Optimizing the Probe Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24
Using Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-27
Section 3.2 Selecting a Data Analysis Approach and Determining Results . . 3-31
Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-32
Relative or Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-32
Relative Standard Curve Method for Quantification . . . . . . . . . . . . . . . . . . . . . . 3-36
Comparative CT Method for Relative Quantification. . . . . . . . . . . . . . . . . . . . . . 3-42
Comparative CT Method for Relative Quantification. . . . . . . . . . . . . . . . . . . . . . 3-45
Standard Curve Method for Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . 3-53

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Chapter 3 Gene Expression and Other Quantitative Assays

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 Section 3.1 Introduction

Section 3.1 Introduction

In This Section This section covers:

About Quantification Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
Selecting a Quantification Assay Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6
Purchasing a Preformulated or Custom-Designed Quantification Assay . . . . . . . . 3-7
TaqMan Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7
Custom TaqMan Gene Expression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9
Designing Your Own Quantification Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10
Primer and Probe Design Using Primer Express Software . . . . . . . . . . . . . 3-11
Selecting the Appropriate Reagent Configuration . . . . . . . . . . . . . . . . . . . 3-14
Using the Universal Thermal Cycling Parameters . . . . . . . . . . . . . . . . . . . 3-16
Optimizing Primer Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21
Optimizing the Probe Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-24
Using Multiplex PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-27

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Chapter 3 Gene Expression and Other Quantitative Assays

About Quantification Assays

What Is a A quantification assay is a real-time PCR assay that measures the amount of a
Quantification nucleic acid target during each amplification cycle of the PCR. The target can be
Assay? DNA, cDNA, or RNA.
Three types of quantification assays are discussed in this chemistry guide:
• DNA/cDNA quantification
• RNA quantification using one-step reverse transcription polymerase chain
reaction (RT-PCR)
• RNA quantification using two-step RT-PCR
Note: For more information on one-step and two-step RT-PCR, see “About Real-
Time PCR Assays” on page 1-4.

Instruments Quantification Assays can be used with the following instruments:

• Applied Biosystems 7900HT Fast Real-Time PCR System (7900HT System)
• ABI PRISM® 7900HT Sequence Detection System, upgradeable to the Applied
Biosystems 7900HT Fast Real-Time PCR System with the 7900HT System Fast
Service Upgrade
• Applied Biosystems 7300 Real-Time PCR System (7300 System)
• Applied Biosystems 7500 Real-Time PCR System (7500 System), upgradeable
to the Applied Biosystems 7500 Fast Real-Time PCR System with the 7500 Fast
Real-Time PCR Upgrade Kit
• Applied Biosystems 7500 Fast Real-Time PCR System (7500 Fast System)

Terms Used in
Quantification Table 3-1 Terms used in quantification analysis
Analysis
Term Definition

Amplicon A short segment of DNA amplified during PCR.

Amplification plot The graphical display of fluorescence signal versus cycle

number.

Baseline The initial cycles of PCR, in which there is little change in

fluorescence signal.

Threshold A level of delta Rnthat is automatically determined by the

Sequence Detection Systems software or manually set and
that is used for CT determination in real-time assays. The level
is set to be above the baseline and sufficiently low to be within
the exponential growth region of the amplification curve. The
threshold is the line whose intersection with the Amplification
plot defines the CT.

Threshold cycle (CT) The fractional cycle number at which the fluorescence passes
the threshold.

Calibrator A sample used as the basis for comparative results.

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 About Quantification Assays

Table 3-1 Terms used in quantification analysis (continued)

Term Definition

Endogenous control Internal control gene present in each experimental sample.

By using an endogenous control as an active reference, you
can normalize quantification of a messenger RNA (mRNA)
target for differences in the amount of total RNA added to each
reaction.

Intron A non-coding segment of a gene.

No template control A sample that does not contain template. It is used to verify
(NTC) amplification quality.

Nucleic acid target (also Nucleotide sequence that you want to detect or quantify.
called “target
template”)

Passive reference A dye that provides an internal fluorescence reference to which

the reporter dye signal can be normalized during data analysis.
Normalization is necessary to correct for fluorescent
fluctuations caused by changes in concentration or volume. A
passive reference dye is included in all real-time PCR reagent
kits.

Reporter dye The dye attached to the 5′ end of a TaqMan® probe.

The dye provides a signal that is an indicator of specific
amplification.

Normalized reporter The ratio of the fluorescence emission intensity of the reporter
(Rn) dye to the fluorescence emission intensity of the passive
reference dye.

Delta Rn (∆Rn) The magnitude of the signal generated by the specified set of
PCR conditions.
The ∆Rn value is determined by the formula:
Rn – baseline

Standard A sample of known concentration used to construct a standard

curve.
By running standards of varying concentrations, you create a
standard curve from which you can extrapolate the quantity of
an unknown sample. See “Relative Standard Curve Method for
Quantification” on page 3-36.

Unknown sample A sample containing an unknown quantity of template that you

want to characterize.

Figure 3-1 shows a representative amplification plot and includes some of the terms
defined above.

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Chapter 3 Gene Expression and Other Quantitative Assays

Rn+

Sample
∆Rn

Rn
Threshold
Rn-
No Template Control
Baseline

CT Cycle

Figure 3-1 Model of a single-sample amplification plot, showing terms

commonly used in quantitative analysis

How Real-Time Real-time PCR allows reactions to be characterized by the point in time during
PCR Quantifica- cycling when amplification of a PCR product achieves a fixed level of fluorescence,
tion Assays Work rather than the amount of PCR product accumulated after a fixed number of cycles.
An amplification plot graphically displays the fluorescence detected over the number
of cycles that were performed.
As shown in Figure 3-1, in the initial cycles of PCR, there is no significant change in
fluorescence signal. This predefined range of PCR cycles is called the “baseline”.
First, the software generates a baseline subtracted amplification plot by calculating a
mathematical trend using Rn values corresponding to the baseline cycles. Then, an
algorithm searches for the point on the amplification plot at which the delta Rn value
crosses the threshold. The fractional cycle at which this occurs is defined as the CT.

Selecting a Quantification Assay Chemistry

Chemistries Quantification assays can be used with the following chemistries:
• TaqMan® Probe-based fluorogenic 5′ nuclease chemistry
• SYBR® Green I dye chemistry
Both TaqMan Probe-based and SYBR Green I dye chemistries can be used for either
one-step or two-step RT-PCR.

Primers Used for For one-step RT-PCR, sequence-specific reverse primers can be used for cDNA
cDNA Synthesis synthesis.
in One-Step Note: Although one-step RT-PCR offers the convenience of a single-tube
RT-PCR preparation for RT and PCR amplification, AmpErase® UNG cannot be used with
this method.

Primers Used for For two-step RT-PCR, the following primers can be used for cDNA synthesis:
cDNA Synthesis • Oligo d(T)16
in Two-Step
• Random hexamers
RT-PCR

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 Purchasing a Preformulated or Custom-Designed Quantification Assay

• Sequence-specific reverse primers

The choice of primers for reverse transcription is best made after experimentally
evaluating all three priming systems. For short RNA sequences containing no hairpin
loops, any of the three priming systems works equally well. For longer RNA
transcripts or sequences containing hairpin loops, consider the guidelines in
Table 3-2.

Table 3-2 Priming systems for RT-PCR

Primers Selection Guidelines

Oligo d(T)16 • Use to reverse transcribe only eukaryotic mRNAs and

retroviruses with poly-A tails
• Avoid long mRNA transcripts or amplicons greater than
2 kilobases upstream

Random primers • Try first for use with long reverse transcripts or reverse
transcripts containing hairpin loops
• Use to transcribe all RNA (rRNA, mRNA, and tRNA)

Sequence-specific • Use to reverse transcribe RNA-containing complementary

reverse primers sequences only
• Use in one-step reactions

Purchasing a Preformulated or Custom-Designed

Quantification Assay

TaqMan Gene Expression Assays

Product TaqMan® Gene Expression Assays are a comprehensive collection of predesigned

Description primer and probe sets, which help researchers quickly and easily perform quantitative
gene expression studies on human, mouse, rat, Arabidopsis, and Drosophila genes.

Product TaqMan Gene Expression Assays are built on Applied Biosystems 5′ nuclease
Properties chemistry. Each assay consists of two unlabeled PCR primers and a FAM™ dye-
labeled TaqMan® MGB (minor groove binder) probe. All components are quality
control-tested and formulated as a single 20✕ mix.
TaqMan Gene Expression Assays are designed to
• Run under universal conditions for two-step RT-PCR.

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Chapter 3 Gene Expression and Other Quantitative Assays

• Work with either of two master mixes: TaqMan® 2✕ Universal PCR Master Mix
(with or without AmpErase ® UNG) or TaqMan® Fast Universal PCR Master
Mix (2✕), No AmpErase® UNG.
Note: The TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG,
is supported only in the 7500 Fast System and Fast-capable 7900HT Systems.
The Fast-capable 7900HT Systems are the Applied Biosystems 7900HT Fast
Real-Time PCR System or the ABI PRISM® 7900HT Sequence Detection
System upgraded to an Applied Biosystems 7900HT Fast Real-Time PCR
System with the 7900HT System Fast Service Upgrade.
• Amplify target cDNA without amplifying genomic DNA (m suffix in assay ID),
when possible. This is achieved by designing probes that cross exon-exon
junctions.

Available TaqMan TaqMan Gene Expression Assays (PNs 4331182 and 4351372) are available for
Gene Expression human, mouse, rat, Arabidopsis, and Drosophila genes. Additionally, a number of
Assays TaqMan® Endogenous Controls are available for all species, with either VIC® dye-
labeled or FAM™ dye-labeled TaqMan® MGB probes. TaqMan Endogenous Controls
with VIC dye labels are primer-limited.
The prefix of the assay name indicates the species for which the assay was designed:
Hs for Homo sapiens (human), Mm for Mus musculus (mouse), Rn for Rattus
norvegicus (rat), At for Arabidopsis thaliana, and Dm for Drosophila melanogaster.
The suffix of the assay name indicates the assay placement, as described in the table
below.

Suffix Description

_m The assay’s probe spans an exon junction and will not detect genomic DNA.

_s The assay’s primers and probes are designed within a single exon and will
detect genomic DNA.

_g The assay may detect genomic DNA.

_mH The assay was designed to a transcript belonging to a gene family with high
sequence homology. The assay provides between 10 CT and 15 CT difference
_sH between the target gene and the gene with the closest sequence homology.
This means the assay will detect the target transcript with 1000- to 3000-fold
_gH greater discrimination (sensitivity) than the closest homologous transcript, if
they are present at the same copy number in a sample.

Note: TaqMan Gene Expression Assays labeled with Hs999999xx_yy are converted
TaqMan® Pre-Developed Assay Reagents (PDAR) designs; these do not follow the
same design rules listed above.
The latest information on available products and specific product uses can be found
on the Applied Biosystems Web site:
http://www.allgenes.com

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 Purchasing a Preformulated or Custom-Designed Quantification Assay

Custom TaqMan Gene Expression Assays

Product If a particular gene expression primer and probe set of interest is not available as a
Description product on the Applied Biosystems Web site, you can use our Custom TaqMan®
Gene Expression Assay Service to submit sequences for any species, gene, or splice
variant. See “Custom TaqMan SNP Genotyping and Gene Expression Assays” on
page 1-7 for additional details on Custom TaqMan® Assays. More information about
Custom TaqMan Gene Expression Assays and related products, part numbers, and
free software is also available at:
http://www.allgenes.com
To place an order, contact your Applied Biosystems representative.

Product Each Custom TaqMan Gene Expression Assay is manufactured based solely on the
Properties customer’s input target sequence. All information supplied by the customer and
returned by Applied Biosystems to the customer is considered confidential and is
treated accordingly. Visit the Applied Biosystems support web site to access tutorials
on how to submit sequences to the Custom TaqMan® Gene Expression Assay
Service. See “How to Obtain Support” on page ix.

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Chapter 3 Gene Expression and Other Quantitative Assays

Designing Your Own Quantification Assay

Important Design This section discusses the Applied Biosystems assay design guidelines that contain
Steps the following important steps:
• Designing primers and probes using Primer Express® software
• Selecting the appropriate reagent configuration (TaqMan® Universal PCR
Master Mix or SYBR® Green PCR Master Mix)
• Using universal thermal cycling parameters
• Using default primer and probe concentrations (or optimizing, if necessary)
IMPORTANT! These steps provide a rapid and reliable system for assay design and
optimization only when used in their entirety. The system must be adopted as a whole
in order to achieve the highest level of success, due to the interdependence of many
of the individual components.

Conclusions The Applied Biosystems Assay Design Guidelines enable Quantitative Assays to be
designed and optimized rapidly and efficiently. Since thousands of assays have been
developed this way, the following conclusions can be made.
• For the vast majority of 5′ nuclease quantification assays designed and run
following these guidelines, using a concentration of 900-nM primers and
250-nM probe provides for a highly reproducible and sensitive assay when using
DNA or cDNA as a template.
• Due to the nonspecific nature of its detection, SYBR Green I dye primer
optimization should be bypassed only with caution. However, if all guidelines
are followed, concentrations of 50-nM forward and reverse primer should
provide robust amplification with a good level of specificity when using DNA
or cDNA as a template. This assumption should, however, always be verified by
checking for nonspecific product formation with either dissociation curve or gel
analysis.
• As a general rule, a 5′ nuclease quantification assay should enable detection and
accurate quantification down to less than 50 copies of a target sequence, with
even greater sensitivity possible.
• A SYBR Green I dye Quantification Assay is capable of similar performance;
however, nonspecific product formation can potentially increase the minimum
detection limit.

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 Designing Your Own Quantification Assay

Primer and Probe Design Using Primer Express Software

The Primer Express® software uses recommended parameters to select primers and
probes based on the DNA sequence that you provide.
If you are designing your own assay, follow the summary of the primer and probe
design guidelines for Quantitative Assays shown in Table 3-3 on page 3-13. For a
detailed discussion of these guidelines see “About the Primer and Probe Design
Guidelines” below.
Note: Even though a probe is not required for SYBR Green I dye detection, it is still
a good idea to use Primer Express software to select a primer and probe set when
designing a SYBR Green I dye assay. Although no probe will be used, the primers
will meet all the required criteria and if, in the future, there is the need to convert the
assay to 5′ nuclease assay chemistry to obtain higher specificity, the probe can
immediately be found in the original Primer Express software document.

Selecting an Selecting a good amplicon site ensures amplification of the target mRNA/cDNA
Amplicon Site for without co-amplifying the genomic sequence, pseudogenes, and other related genes.
Gene Expression SYBR Green I dye chemistry can be useful for screening amplicon sites for gene
Assays expression.

Guidelines
• The amplicon should span one or more introns to avoid amplification of the
target gene in genomic DNA.
• The primer pair should be specific to the target gene to avoid amplification of
pseudogenes or other related genes.
• When designing primers, use Primer Express software guidelines.
• If no good sequence is found, it may be necessary to examine the sequence and
redesign the amplicon or simply screen for more sites.
If the gene you are studying does not have introns, then it is not possible to design an
amplicon that will amplify the mRNA sequence without amplifying the gene
sequence. In this case, it is necessary to run RT minus controls.

About the Primer Selection of Small Amplicons

and Probe Design An important default parameter in Primer Express software is the selection of
Guidelines amplicons in the 50- to 150-base pair range. Small amplicons are favored because
they promote high-efficiency amplification.
In addition, high-efficiency assays enable relative quantification to be performed
using the comparative CT method ( ∆∆C T ) (Livak and Schmittgen, 2001). This
method increases sample throughput by eliminating the need for standard curves
when looking at expression levels of a target relative to a calibrator sample. (For
more information on the comparative CT method, see page 3-40.)

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Chapter 3 Gene Expression and Other Quantitative Assays

G/C Content
Whenever possible, primers and probes should be selected in a region with a G/C
content of 30 to 80%. Regions with a G/C content in excess of this may not denature
well during thermal cycling, leading to a less efficient reaction. In addition, G/C-rich
sequences are susceptible to nonspecific interactions that may reduce reaction
efficiency and produce nonspecific signal in SYBR Green I dye assays. For this same
reason, primer and probe sequences containing runs of four or more G bases should
be avoided.

Melting Temperature
Selecting primers and probes with the recommended melting temperature (Tm)
allows the use of universal thermal cycling parameters. Having the probe Tm be
10 °C higher than that of the primers is recommended.

5′ End of Probes
Primer Express software does not select probes with a G on the 5′ end. The
quenching effect of a G base in this position will be present even after probe
cleavage. This can result in reduced fluorescence values ( ∆Rn , see Table 3-1 on
page 3-4), which can impact the performance of an assay. Having G bases in
positions close to the 5′ end, but not on it, has not been shown to compromise assay
performance.

3′ End of Primers
The last five bases on the 3′ end of the primers should contain no more than two C
and/or G bases, which is another factor that reduces the possibility of nonspecific
product formation. Under certain circumstances, such as a G/C-rich template
sequence, this recommendation may have to be relaxed to keep the amplicon under
150 basepairs in length. In general, avoid primer 3′ ends extremely rich in G and/or C
bases.

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 Designing Your Own Quantification Assay

Summary of
Primer and MGB Table 3-3 Primer and probe design guidelines for quantitative assays
Probe Design
Guidelines Probe Guidelines Primer Guidelines

Select the probe first, then design the primers as close as possible to the probe without
overlapping the probe (amplicons of 50 to 150 basepairs are strongly recommended).

Keep the G/C content in the 30 to 80% range.

Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs
should be avoided.

When using Primer Express software, the When using Primer Express software, the
Tm should be 68 to 70 °C. Tm should be 58 to 60 °C.

No G on the 5′ end. The five nucleotides at the 3′ end should

have no more than two G and/or C bases.
Make TaqMan MGB probes as short as
possible without being shorter than
13 nucleotides.

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Chapter 3 Gene Expression and Other Quantitative Assays

Selecting the Appropriate Reagent Configuration

Several TaqMan® Probe-based and SYBR® Green I dye chemistry kits are available
for quantitative assays. The reagent configuration you use depends on your assay
type (Table 3-4, Table 3-5, Table 3-6).

Recommended Note: See Appendix B for a list of available kit sizes.

Reagent
Configurations Table 3-4 Reagents available for DNA and cDNA quantification assays

Chemistry Reagent Configuration Part Number

TaqMan probes TaqMan Fast Universal PCR Master Mix (2✕), No 4352042
AmpErase® UNG, 250 Reactions a

TaqMan 2✕ Universal PCR Master Mix, 200 4304437

Reactions

TaqMan 2✕ Universal PCR Master Mix, 2000 4326708

Reactions

10-Pack, TaqMan 2✕ Universal PCR Master Mix 4305719

TaqMan 2✕ Universal PCR Master Mix, No 4324018

AmpErase UNG, 200 Reactions

TaqMan 2✕ Universal PCR Master Mix, No 4326614

AmpErase UNG, 2000 Reactions

10-Pack, TaqMan 2✕ Universal PCR Master Mix, 4324020

No AmpErase UNG

TaqMan PCR Core Reagents Kit, 200 Reactions N808-0228

SYBR Green I dye SYBR Green PCR Master Mix, 200 reactions 4309155

SYBR Green PCR Master Mix, 50-mL (2000 4334973

reactions)

SYBR Green PCR Master Mix, 1-mL (40 reactions) 4344463

SYBR Green PCR Core Reagents, 200 Reactions 4304886

a. The TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, can be used with the
7500 Fast System, the 7500 System upgraded to a 7500 Fast System, and with Fast-
capable 7900HT Systems (the Applied Biosystems 7900HT Fast Real-Time PCR System or
the ABI PRISM® 7900HT Sequence Detection System upgraded to an Applied Biosystems
7900HT Fast Real-Time PCR System with the 7900HT System Fast Service Upgrade).

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 Designing Your Own Quantification Assay

RNA Quantification Using One-Step RT-PCR

Table 3-5 Reagents available for RNA quantification assays using one-step
RT-PCR

Chemistry Reagent Configuration Part Number

TaqMan probes TaqMan One-Step RT-PCR Master Mix Reagents 4309169

Kit

TaqMan Gold RT-PCR Kit (without controls) N808-0232

TaqMan EZ RT-PCR Core Reagents N808-0236

Note: Use this configuration when a high-
temperature RT step is required.

SYBR Green I dye SYBR Green RT-PCR Reagents 4310179

RNA Quantification Using Two-Step RT-PCR

Table 3-6 Reagents available for RNA quantification assays using two-step
RT-PCR

Chemistry Step Reagent Configuration Part Number

TaqMan PCR step only TaqMan 2✕ Universal PCR Master 4304437

probes Mix

TaqMan Fast Universal PCR Master 4352042

Mix (2✕), No AmpErase UNG a

RT step only TaqMan Reverse Transcription N808-0234

Reagents

High-Capacity cDNA Archive Kit 4322171

Both RT and TaqMan Gold RT PCR kit N808-0232

PCR steps
TaqMan EZ RT-PCR Core Reagents N808-0236
Without Controls

TaqMan EZ-RT-PCR Core Reagents, 403028

10-pack

SYBR Green I PCR step only SYBR Green Master Mix 4309155
dye
Both RT and SYBR Green RT-PCR Reagents 4310179
PCR steps

a. The TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, can be used with the
7500 Fast System, the 7500 System upgraded to a 7500 Fast System, and with Fast-
capable 7900HT Systems (the Applied Biosystems 7900HT Fast Real-Time PCR System or
the ABI PRISM® 7900HT Sequence Detection System upgraded to an Applied Biosystems
7900HT Fast Real-Time PCR System with the 7900HT System Fast Service Upgrade).

About Master Mix TaqMan 2✕ Universal PCR Master Mix

Reagents The TaqMan 2✕ Universal PCR Master Mix is designed to provide optimal

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Chapter 3 Gene Expression and Other Quantitative Assays

performance for 5′ nuclease assays that use cDNA or DNA as a template. This
product contains components that ensure excellent assay performance even when
demanding G/C-rich target sequences are encountered. The use of one reagent for all
assays simplifies the process of assay implementation.

TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG

Use the TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG, to run
gene expression assays on the Applied Biosystems 7900HT Fast Real-Time PCR
System in about 35 minutes and on the Applied Biosystems 7500 Fast Real-Time
PCR System in about 40 minutes.

SYBR Green I PCR Master Mix

The SYBR Green PCR Master Mix is a convenient premix for real-time PCR using
the SYBR Green I dye. Direct detection of the PCR product is monitored by
measuring the increase in fluorescence that is caused by the SYBR Green I dye
binding to double-stranded DNA.
Note: You cannot use SYBR Green I dye with the TaqMan Fast Universal PCR
Master Mix (2✕), No AmpErase UNG.

About the AmpliTaq Gold DNA Polymerase

Reagent The use of the hot-start enzyme AmpliTaq Gold® DNA Polymerase is an integral part
Components of Applied Biosystems assay design guidelines for both TaqMan Probe-based and
SYBR Green I dye chemistries. The use of AmpliTaq Gold DNA Polymerase ensures
a robust reaction and can dramatically reduce the amount of nonspecific product
formation. A further benefit is the simplification of assay setup, which can be
performed at room temperature.
Note: The DNA polymerase system included in the TaqMan Fast Universal PCR
Master Mix (2✕), No AmpErase UNG, is capable of instant hot-start PCR and does
not require an activation step. The performance is similar to that of the AmpliTaq
Gold DNA Polymerase.

MultiScribe Reverse Transcriptase

MultiScribe™ Reverse Transcriptase is a recombinant Moloney Murine Leukemia
Virus (MuLV) Reverse Transcriptase.

Using the Universal Thermal Cycling Parameters

All quantitative assays designed using Applied Biosystems assay design guidelines
can be run using the universal thermal cycling parameters. This protocol design
eliminates any optimization of the thermal cycling parameters and means that
multiple assays can be run on the same plate without sacrificing performance. This
benefit is critical when combining two assays into a multiplex 5′ nuclease assay
system.

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 Designing Your Own Quantification Assay

Recommended DNA/cDNA Quantification

Thermal Cycling The thermal cycling parameters listed in Table 3-7 are recommended for DNA and
Parameters cDNA Quantification Assays. The parameters apply to both TaqMan Probe-based
and SYBR Green I dye chemistries.

Table 3-7 Thermal cycling parameters for DNA and cDNA quantification assays
using TaqMan 2✕ Universal PCR Master Mix

Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpliTaq Gold
AmpErase UNG
DNA Polymerase Melt Anneal/Extend
Activation
Activation

HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

Table 3-8 Thermal cycling parameters for DNA and cDNA quantification assays
using TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG for the
7900HT Fast System

Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpErase UNG
Denature Melt Anneal/Extend
Activation a

HOLD HOLD CYCLE

2 min @ 50 °C 20 sec @ 95 °C 1 sec @ 95 °C 20 sec @ 60 °C

a. Required only if AmpErase UNG is added to the reactions.

Table 3-9 Thermal cycling parameters for DNA and cDNA quantification assays
using TaqMan Fast Universal PCR Master Mix (2✕), No AmpErase UNG for the
7500 Fast System

Times and Temperatures

Initial Steps PCR (40 Cycles)

AmpErase UNG
Denature Melt Anneal/Extend
Activation a

HOLD HOLD CYCLE

2 min @ 50 °C 20 sec @ 95 °C 3 sec @ 95 °C 30 sec @ 60 °C

a. Required only if AmpErase UNG is added to the reactions.

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Chapter 3 Gene Expression and Other Quantitative Assays

RNA Quantification Using One-Step RT-PCR

The thermal cycling parameters listed in Table 3-10 are recommended for RNA
quantification assays using one-step RT-PCR. The parameters apply to both TaqMan
Probe-based and SYBR Green I dye chemistries.

Table 3-10 Thermal cycling parameters for RNA quantification assays using
one-step RT-PCR

Times and Temperatures a

Initial Steps PCR (40 Cycles)

AmpliTaq Gold
Reverse
DNA Polymerase Melt Anneal/Extend
Transcription
Activation

HOLD HOLD 40 CYCLES

30 min @ 48 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

a. Not applicable for the TaqMan EZ RT-PCR Kit Protocol. See the TaqMan EZ RT-PCR Kit
Protocol for the appropriate values.

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 Designing Your Own Quantification Assay

RNA Quantification Using Two-Step RT-PCR

The thermal cycling parameters listed in Tables 3-11 through 3-14 are recommended
for RNA quantification assays using two-step RT-PCR. The parameters apply to both
TaqMan Probe-based and SYBR Green I dye chemistries.

Table 3-11 Thermal cycling parameters for RNA quantification assays using
two-step RT-PCR on the 7900HT System

Step Times and Temperatures

HOLD a HOLD HOLD —

1. RT Step
10 min @ 25 °C 30 min @ 48 °C 5 min @ 95 °C

Initial Steps PCR (40 Cycles)

2. PCR Step AmpliTaq Gold

Using AmpErase UNG DNA Anneal/
TaqMan 2✕ Melt
Activation Polymerase Extend
Universal Activation
PCR Master
Mix HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

2. PCR Step Initial Steps PCR (40 Cycles)

Using
TaqMan Fast AmpErase UNG Anneal/
Universal Denature Melt
Activation b Extend
PCR Master
Mix (2✕), No HOLD HOLD CYCLE
AmpErase
UNG 2 min @ 50 °C 20 sec @ 95 °C 1 sec @ 95 °C 20 sec @ 60 °C

a. This step is required for random hexamers or oligod(T). It is not necessary when using
sequence-specific primers.
b. Required only if AmpErase UNG is added to the reactions.

Table 3-12 Thermal cycling parameters for RNA quantification assays using
two-step RT-PCR on the 7500 Fast System

Step Times and Temperatures

HOLD a HOLD HOLD —

1. RT Step
10 min @ 25 °C 30 min @ 48 °C 5 min @ 95 °C

Initial Steps PCR (40 Cycles)

2. PCR Step AmpliTaq Gold

Using AmpErase UNG DNA Anneal/
TaqMan 2✕ Melt
Activation Polymerase Extend
Universal Activation
PCR Master
Mix HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

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Chapter 3 Gene Expression and Other Quantitative Assays

Table 3-12 Thermal cycling parameters for RNA quantification assays using
two-step RT-PCR on the 7500 Fast System (continued)

Step Times and Temperatures

2. PCR Step Initial Steps PCR (40 Cycles)

Using
TaqMan Fast AmpErase UNG Anneal/
Universal Denature Melt
Activation b Extend
PCR Master
Mix (2✕), No HOLD HOLD CYCLE
AmpErase
UNG 2 min @ 50 °C 20 sec @ 95 °C 3 sec @ 95 °C 30 sec @ 60 °C

a. This step is required for random hexamers or oligod(T). It is not necessary when using
sequence-specific primers.
b. Required only if AmpErase UNG is added to the reactions.

Table 3-13 Thermal cycling parameters for RNA Quantification assays using the
high-capacity cDNA archive kit when using two-step RT-PCR on the 7900HT
System

Step Times and Temperatures

HOLD HOLD

1. RT Step Step 1 Step 2

10 min @ 25 °C 120 min @ 37 °C

Initial Steps PCR (40 Cycles)

2. PCR Step AmpliTaq Gold

AmpErase
Using TaqMan DNA Anneal/
UNG Melt
2✕ Universal Polymerase Extend
Activation
PCR Master Activation
Mix
HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

2. PCR Step Initial Steps PCR (40 Cycles)

Using TaqMan
Fast Universal AmpErase
PCR Master Anneal/
UNG Denature Melt
Mix (2✕), No Extend
Activation a
AmpErase
UNG
HOLD HOLD CYCLE

2 min @ 50 °C 20 sec @ 95 °C 1 sec @ 95 °C 20 sec @ 60 °C

a. Required only if AmpErase UNG is added to the reactions.

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 Designing Your Own Quantification Assay

Table 3-14 Thermal cycling parameters for RNA Quantification assays using the
high-capacity cDNA archive kit when using two-step RT-PCR on the 7500 Fast
System

Step Times and Temperatures

HOLD HOLD

1. RT Step Step 1 Step 2

10 min @ 25 °C 120 min @ 37 °C

Initial Steps PCR (40 Cycles)

2. PCR Step AmpliTaq Gold

AmpErase
Using TaqMan DNA Anneal/
UNG Melt
2✕ Universal Polymerase Extend
Activation
PCR Master Activation
Mix
HOLD HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 95 °C 1 min @ 60 °C

2. PCR Step Initial Steps PCR (40 Cycles)

Using TaqMan
Fast Universal AmpErase
PCR Master Anneal/
UNG Denature Melt
Mix (2✕), No Extend
Activation a
AmpErase
UNG
HOLD HOLD CYCLE

2 min @ 50 °C 20 sec @ 95 °C 3 sec @ 95 °C 30 sec @ 60 °C

a. Required only if AmpErase UNG is added to the reactions.

IMPORTANT! For most applications and when large amounts of cDNA are required,
Applied Biosystems recommends 120 minutes at 37 °C for reverse transcription to
achieve optimal conversion.

Optimizing Primer Concentrations

By independently varying forward and reverse primer concentrations, you can
identify the concentrations that provide optimal assay performance. Primers are
always in large molar excess during the exponential phase of PCR amplification; by
adjusting their initial concentration, their effective melting temperatures can be
adjusted.
When using the TaqMan 2✕ Universal PCR Master Mix, Applied Biosystems
recommends the primer concentrations shown in Table 3-15. Detailed discussions
follow for the:
• Primer optimization matrix (page 3-22)
• 5′ Nuclease quantification assays (using TaqMan Probe-based chemistry, page
3-22)
• SYBR Green I Dye quantification assays (page 3-23)

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Chapter 3 Gene Expression and Other Quantitative Assays

Default Primer The recommended primer concentrations listed in Table 3-15 are for DNA and
Concentrations cDNA quantification assays.

Table 3-15 Recommended primer concentrations for DNA and cDNA

quantification assays

Concentrations (nM)
Chemistry
Forward Primer Reverse Primer

TaqMan probe 900 900

SYBR Green I dye 50 50

Primer A primer optimization matrix allows you to determine the minimum primer
Optimization concentration yields the minimum CT and maximum ∆Rn.
Matrix A primer optimization matrix can help to compensate for nonspecific primer
binding, which can reduce the amount of primer available to bind at its specific site.

5′ Nuclease For a 5′ nuclease quantification assay, optimal performance is achieved by selecting

Quantification the primer concentrations that provide the lowest CT and highest ∆Rn for a fixed
Assays amount of target template.
The results of a typical TaqMan primer optimization matrix experiment are shown in
Figure 3-2. Figure 3-2(a) shows the amplification plots for all primer concentration
combinations in linear view. Figure 3-2(b) shows the same data in log view format.
The combination of 50-nM forward and reverse primer (Plot group C) gives both the
lowest ∆Rn and highest CT. All other primer combinations that contain a 150-nM
concentration of either the forward or reverse primer (Plot group B) give a reduced
∆Rn . All primer combinations that contain at least 300-nM forward and reverse
primer (Plot group A) give both the highest ∆Rn and the lowest CT; as a result, any of
the plot group A or B would provide optimal performance.
It should be noted that, although CT values are the parameter by which quantitative
values are assigned in a real-time Quantification Assay, ∆Rn values can also prove
important when trying to obtain maximum sensitivity and reproducibility.

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 Designing Your Own Quantification Assay

a) Linear view

} Plot Group A

} Plot Group B

∆Rn
} Plot Group C

Cycle

b) Log view
} Plot Group A
} Plot Group B

} Plot Group C
∆Rn

Cycle

Figure 3-2 Primer optimization experimental results showing amplification plots

of primer combinations
Plot group key:
A: Combinations that contain at least 300 nM of forward and reverse primer
B: Combinations that contain at least 150 nM of forward and reverse primer
C: Combinations that contain at least 50 nM of forward and reverse primer

SYBR ® Green I Optimizing primer concentrations is slightly more complex for a SYBR Green I dye
Dye Quantifica- quantification assay. The same primer optimization matrix should be performed;
tion Assays however, this time it must include NTCs. In this case, the primer concentrations
selected should provide a low CT and high ∆Rn when run against the target template,
but should not produce nonspecific product formation with NTCs. An ideal NTC
amplification plot is shown in Figure 3-1 on page 3-6.
Dissociation curves or gel analysis can be extremely useful when selecting optimal
primer concentrations for a SYBR Green I dye Quantification Assay. This is
demonstrated in Figure 3-3 on page 3-24, which shows the results from a primer
optimization matrix at primer concentrations of 900-nM forward and reverse
primers. The strong amplification of the NTC wells shown in Figure 3-3(a) indicates
that significant nonspecific amplification is occurring. This is confirmed by the
dissociation curve data shown in Figure 3-3(b), which shows that the melting

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Chapter 3 Gene Expression and Other Quantitative Assays

temperature of the product generated in the absence of template is lower than the
melting temperature of the specific product generated with template. This is typical
of primer-dimer formation and indicates that lower primer concentrations may
provide more optimal results.

Target Amplification

NTC (nonspecific
amplification)

Target
NTC Amplification
(non-specific
amplification)

Figure 3-3 Amplification data using SYBR Green I dye chemistry

(a) Amplification plot (linear view) demonstrating suspected nonspecific
amplification in NTC wells.
(b) Dissociation curve analysis confirming that product in NTC wells has a
different melting temperature from the specific product.

Optimizing the Probe Concentration

The recommended probe concentration of 250 nM ensures excellent assay
performance. However, depending on the requirements of the assay, a probe
optimization experiment can prove useful.
Note: No probe is required for SYBR Green I dye detection.

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 Designing Your Own Quantification Assay

Recommended The recommended probe concentrations for DNA and cDNA quantification assays
Probe using TaqMan Probe-based chemistry is 250 nM.
Concentrations Figure 3-4 shows the results of a probe optimization experiment in which the probe
concentration is varied from 50 to 250 nM. Figure 3-4(a) shows an increase in ∆Rn
as the probe concentration is increased, whereas Figure 3-4(b) shows that the CT
value changes with sufficient probe concentrations.
It should be noted, however, that to ensure the best reproducibility, especially when
wishing to detect low copy numbers of a target sequence, it is necessary to avoid
probe limiting concentrations. The assay should be run at a probe concentration of
250 nM. By using a 250 nM concentration, probe limitation is avoided and large ∆Rn
values are ensured. Large ∆Rn values indicate a robust assay that is performing at
high efficiency, giving high product yield and allowing more accurate peak
measurement.

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Chapter 3 Gene Expression and Other Quantitative Assays

a) Linear
view 250 nM P robe

150 nM P robe
∆Rn

100 nM P robe

50 nM P robe

C ycle
b) Log view

250 nM Probe
150 nM Probe
100 nM Probe
50 nM Probe
∆Rn

Cycle

Figure 3-4 Amplification plot (linear and log views) of probe concentration
titration from 50 to 250 nM

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 Designing Your Own Quantification Assay

Using Multiplex PCR

Multiplex PCR is the use of more than one primer/probe set in the same tube.
Multiplex PCR is most commonly used in 5′ nuclease assays for the relative
quantification of gene expression.
Typically one probe (labeled with FAM™ dye) is used to detect the target species;
another probe (labeled with VIC® dye) is used to detect an endogenous control
(internal control gene). Running both assays in a single tube reduces both the running
costs and the dependence on accurate pipetting when splitting a sample into two
separate tubes.

Multiplex in In order to multiplex using the comparative CT method, you must ensure that the
Contrast to endogenous control that you have selected is more abundant (lower CT) than all of the
Singleplex targets that you are trying to quantify under all conditions and then you must run the
endogenous control assay as a primer-limited assay. For multiplexing, the
endogenous control assay (for the more abundant template) in each reaction must be
primer limited to avoid competitive PCR that may alter the CT of the less abundant
template.
Primer-limited endogenous control reactions are used in multiplex assays to
normalize for the amount of input nucleic acid into different wells as well as to
normalize for effects on the target assay in the same well. However, a primer-limited
assay may be more susceptible to fluctuations in reaction conditions than the primer
non-limited target assay that it is normalizing.
Doing this in a multiplex format becomes increasingly more complex as the number
of targets you wish to quantify increases. It becomes increasingly unlikely that you
will be able to identify a suitable endogenous control that will be more abundant than
all of the targets you wish to query and whose expression does not change as a result
of the experimental conditions or across different samples.
As a result, for an increasing number of targets, it is likely to be more efficient and
effective to run the targets and controls in the singleplex format utilizing the
precision of the real-time PCR system in conjunction with delivery of equivalent
amounts of input material to different reaction wells.
For these reasons, when you analyze multiple numbers of targets it is likely to be
more effective to run your assays in the singleplex format. To multiplex, you would
have to first run all of your target assays and endogenous control assays in both the
multiplex and singleplex format and compare CT values from both formats to
determine if there are any effects of the multiplexing on your CT values—which
could be a larger undertaking than the study itself.
Also by using the singleplex method, any target can potentially be used as an
endogenous control at the analysis step of the process. Any target whose expression
level does not change with experimental conditions or across samples may serve as
an endogenous control. Therefore, the more targets you have in a singleplex format,
the higher the probability that you will have one or more suitable endogenous
controls against which to normalize your remaining targets.

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Chapter 3 Gene Expression and Other Quantitative Assays

Primer Limiting in To generate an accurate multiplex assay, it is important to ensure that the
Multiplex Assays amplification of one species does not dominate the other. Otherwise, the
amplification of a highly abundant species can prevent the less abundant species
from amplifying efficiently. Such a scenario could easily produce inaccurate results
and, in severe cases, inhibit detection of the less abundant species completely. This
situation can be avoided by limiting the concentrations of the primers used to amplify
the more abundant species, thereby “turning off ” the amplification soon after the CT
has been established.
Primer limitation results in the reaction components common to both assays not
being exhausted, allowing the amplification of the less abundant species to continue
at high efficiency. If the more abundant species is not known, it should be determined
before entering into a multiplex assay system by running both targets in separate
tubes. Both amplifications should be primer limited if neither species is consistently
more abundant.

Considering Relative Abundance of the Target and Reference

In applying the primer limitation to target and endogenous control amplifications, the
relative abundance of the two species must be considered. For quantification of gene
expression, it is possible to use rRNA as an endogenous control. The concentration of
rRNA in total RNA is always greater than the concentration of any target mRNA.
Therefore, in multiplex reactions amplifying both target and rRNA, only the
concentrations of the rRNA primers need to be limited.

Limiting Primer Matrix

To define limiting primer concentrations, run a matrix of forward and reverse primer
concentrations using the value of the minimum initial template. The goal is to
identify primer concentrations that reduce the ∆Rn of the assay without affecting the
CT value. Table 3-16 illustrates a recommended matrix of forward and reverse
primers varying in concentration from 20 to 100 nM.
Note: Although following all design criteria does facilitate the ability to identify
limiting primer concentrations, it may not be possible for all assays. If a limiting
primer matrix experiment does not enable the identification of primer limiting
concentrations, it will be necessary to redesign at least one primer or run the
reactions in separate tubes.

Table 3-16 Matrix of varying concentrations of forward and reverse primers (20 to 100 nM)

Forward: 100 nM 100 nM 100 nM 100 nM 100 nM

Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Forward: 80 nM 80 nM 80 nM 80 nM 80 nM
Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Forward: 60 nM 60 nM 60 nM 60 nM 60 nM
Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Forward: 40 nM 40 nM 40 nM 40 nM 40 nM
Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

Forward: 20 nM 20 nM 20 nM 20 nM 20 nM
Reverse: 100 nM 80 nM 60 nM 40 nM 20 nM

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 Designing Your Own Quantification Assay

Example
The results of a limiting primer matrix experiment are shown in Figure 3-5.
Figure 3-5(a) shows that only when lowering the primer concentrations below
approximately 50 nM is the CT value significantly affected. Figure 3-5(b) shows the
corresponding relationship between primer concentrations and ∆Rn , and
demonstrates that lower product yields can be achieved by decreasing forward and
reverse primer concentrations.
The plateau area visible in Figure 3-5(a) shows the region in which suitable primer
limiting concentrations can be found. In this area, the CT (and therefore the
corresponding quantification value) is unchanged, whereas the ∆Rn value and
corresponding product yield are significantly reduced.
For this example, an appropriate selection of primer limiting concentrations would be
at least 50 nM forward and reverse primer. It is important to note that probe
concentration should be kept at an optimal level even when an assay is primer limited
to ensure that the signal produced is large enough for accurate multicomponenting by
the Sequence Detection Systems software.

Figure 3-5 Results from Limiting Primer Matrix experiment

(a) Shows how CT value is affected by variation in forward and reverse primer
concentrations. Plateau region indicated shows area where CT value remains
constant.
(b) Shows reduction in ∆Rn values as primer concentration decreases.

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 Section 3.2 Selecting a Data Analysis Approach and Determining Results

Section 3.2 Selecting a Data Analysis Approach and

Determining Results

In This Section This section covers:

Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-32
Relative or Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-32
Relative Standard Curve Method for Quantification . . . . . . . . . . . . . . . . . . . . . . 3-36
Comparative CT Method for Relative Quantification. . . . . . . . . . . . . . . . . . . . . . 3-42
Comparative CT Method for Relative Quantification. . . . . . . . . . . . . . . . . . . . . . 3-45
Standard Curve Method for Absolute Quantification . . . . . . . . . . . . . . . . . . . . . . 3-53

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Chapter 3 Gene Expression and Other Quantitative Assays

Data Analysis
Data analysis varies depending on the product, assay, and instrument. Refer to the
appropriate instrument user guide for instructions on how to analyze your data.

General Process The general process for analyzing the data from gene expression assays involves:
1. Viewing the amplification plots for the entire plate
2. Setting the baseline and threshold values
3. Using the methods described in this section to determine results

Resources for For more information about analyzing your data, see Livak and Schmittgen, 2001.
Data Analysis Also refer to the following documents:
• The appropriate instrument user guide
• Data Analysis and Relative Quantification chapters in the TaqMan Cytokine
Gene Expression Plate I Protocol (PN 4306744). This protocol provides
examples using multiplex reactions.
• RQ Manager Software User Guide (PN 4351670)
• Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise
Database User Guide (PN 4351684)
• SDS Enterprise Database for the Applied Biosystems 7900HT Fast Real-Time
PCR System Administrator Guide (PN 4351669)
• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative
Quantification Getting Started Guide (PN 4347824)
• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute
Quantification Getting Started Guide (PN 4347825)
• Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation
and Maintenance Getting Started Guide (PN 4347828)
• Applied Biosystems 7500 Fast Real-Time PCR System Performing Fast Gene
Quantification Quick Reference Card (PN 4362285)
• Applied Biosystems 7900HT Fast Real-Time PCR System User Bulletin:
Performing Fast Gene Quantification (PN 4352533)
• TaqMan® Gene Expression Assays Protocol (PN 4333458)
Note: Some documents are available through the Internet (see “How to Obtain
Support” on page ix).

Relative or Absolute Quantification

You can design your assay as relative or absolute quantification. How you calculate
the results of your Quantification Assays depends on your experimental design.

What Is Relative Relative quantification describes the change in expression of the target gene in a test
Quantification? sample relative to a calibrator sample. The calibrator sample can be an untreated
control or a sample at time zero in a time-course study (Livak and Schmittgen, 2001).
Relative quantification provides accurate comparison between the initial level of
template in each sample.

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 Relative or Absolute Quantification

Example
You can compare the level of expression of a gene (Gene A) between a treated and
untreated sample (for example, a drug treatment) relative to the expression of a
second gene (Gene B). The resulting data is the expression of Gene A in treated and
untreated samples normalized to the expression of Gene B.

Calculation Relative quantification can be performed with data from all the real-time PCR
Methods for instruments. The calculation methods used for relative quantification are:
Relative • Relative standard curve method, singleplex PCR
Quantification
• Comparative CT method (∆∆CT), singleplex PCR
• Relative standard curve method, multiplex PCR
• Comparative CT method, multiplex PCR
Note: TaqMan Gene Expression Assays and Custom TaqMan Gene Expression
Assays, as well as assays that use the TaqMan Fast Universal PCR Master Mix (2✕),
No AmpErase UNG, are intended for use in singleplex reactions.

Determining Which Method to Use

Because all the calculation methods can give equivalent results, determine which
method to use by considering the following:
• Relative Standard Curve Method–
Running the target and endogenous control amplifications in separate tubes and
using the relative standard curve method of analysis requires the least amount of
optimization and validation.
• Comparative CT Method
To use the comparative CT method, a validation experiment must be run to show
that the efficiencies of the target and endogenous control amplifications are
approximately equal. The advantage of using the comparative CT method is that
the need for a standard curve is eliminated. This increases throughput because
wells no longer need to be used for the standard curve samples. It also
eliminates the adverse effect of any dilution errors made in creating the standard
curve samples. Here, the target and endogenous control amplifications are run
in separate tubes.
• Multiplex PCR
To amplify the target and endogenous control in the same tube, limiting primer
concentrations must be identified and shown not to affect CT values. By running
the two reactions in the same tube, throughput is increased and the effects of
pipetting errors are reduced.

What Is Absolute Absolute quantification determines the input copy number of the template of interest,
Quantification? usually by relating the PCR signal to a standard curve (Livak and Schmittgen, 2001).

Example
Use absolute quantification to measure viral copy number in samples for which this
information is not known (unknown samples). In order to measure the viral copy
number in the unknown samples, compare the measurement to a standard curve of
known viral copy numbers. Since the basis for measurement is a standard curve with
known quantities of virus, the measurement would be absolute.

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Chapter 3 Gene Expression and Other Quantitative Assays

Calculation Absolute quantification can be performed with data from all the real-time PCR
Methods for instruments. However, the absolute quantities of the standards must first be measured
Absolute by some independent means.
Quantification The calculation method used for absolute quantification is the standard curve
method.

Terms Used The terms in Table 3-17 are used in this discussion of absolute and relative
quantification.

Table 3-17 Terms used in absolute and relative quantification

Control/Term Definition

Standard A sample of known concentration used to construct a standard curve.

Reference A passive or active signal used to normalize experimental results.

Endogenous and exogenous controls are examples of active
references. Active reference means the signal is generated as the
result of PCR amplification. The active reference has its own set of
primers and probe.
• Endogenous control
An RNA or DNA that is in each experimental sample as isolated. By
using an endogenous control as an active reference, you can
normalize quantification of a messenger RNA (mRNA) target for
differences in the amount of total RNA added to each reaction.
• Exogenous control
A characterized RNA or DNA spiked into each sample at a known
concentration. An exogenous active reference is usually an in vitro
construct that can be used as an internal positive control (IPC) to
distinguish true target negatives from PCR inhibition. An exogenous
reference can also be used to normalize for differences in efficiency
of sample extraction or complementary DNA (cDNA) synthesis by
reverse transcriptase.

Passive A dye that provides an internal reference to which the reporter dye
reference signal is normalized.
Whether or not an active reference is used, it is important to use a
passive reference (for example, ROX dye) in order to normalize for non-
PCR-related fluctuations in fluorescence signal.

Normalized A unitless number that can be used to compare the relative amount of
amount of target target in different samples.

CV Coefficient of variation. The ratio of the standard deviation of a

distribution to its arithmetic mean.

Calibrator In relative quantification, the sample used as the basis for comparative
results.

Endogenous Internal control gene present in each experimental sample. By using an

control endogenous control as an active reference, you can normalize
quantification of a messenger RNA (mRNA) target for differences in the
amount of total RNA added to each reaction.

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 Relative or Absolute Quantification

For More For more information on absolute and relative quantification, refer to Livak and
Information Schmittgen, 2001 (see Appendix C on page C-1 for full reference). This document
contains detailed procedures for performing the experiments referenced in this
section.

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Chapter 3 Gene Expression and Other Quantitative Assays

Relative Standard Curve Method for Quantification

It is easy to prepare standard curves for relative quantification because quantity is
expressed relative to some basis sample, such as the calibrator.
For all experimental samples, normalized target quantity is determined from the
standard curve and divided by the normalized target quantity of the calibrator. Thus,
the calibrator becomes the 1✕ sample, and all other quantities are expressed as an n-
fold difference relative to the calibrator. For example, in a study of drug effects on
expression, the untreated control would be an appropriate calibrator.

Requirements Quantification by the relative standard curve method requires that:

• Stock RNA or DNA is accurately diluted. If two-fold dilutions of a total RNA
preparation from a control cell line are used to construct a standard curve, the
units can be the dilution values 1, 0.5, 0.25, 0.125, and so on. By using the same
stock RNA or DNA to prepare standard curves for an entire study, the relative
quantities determined can be compared across the plates.
• For quantification normalized to an endogenous control, standard curves are
prepared for both the target and the endogenous control. For each experimental
sample, the amount of target and endogenous control is determined from the
appropriate standard curve. Then, the target amount is divided by the
endogenous control amount to obtain a normalized target value.
• One of the experimental samples is the calibrator, or 1✕ sample. Each of the
normalized target values is divided by the calibrator normalized target value to
generate the relative expression levels.
• Pipette correctly and accurately. Pipette volumes greater than 5 µL of nucleic
acid sample to minimize inaccuracy.

Endogenous Amplification of an endogenous control can be performed to standardize the amount

Control of sample RNA or DNA added to a reaction. For the quantification of gene
expression, researchers have used ß-actin, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), 18S ribosomal RNA (rRNA), or other transcripts for an
endogenous control.
Another approach is to normalize to a measurement external to the PCR experiment.
For example, you can use UV absorption to determine the amount of RNA added to a
cDNA reaction. You then run a PCR using cDNA derived from the same amount of
input RNA. You can use this approach to select an appropriate endogenous control
and to determine if an endogenous control is affected by the treatment. In this case,
the target gene and the endogenous reference are the same item (Livak and
Schmittgen, 2001).

Standards Because the sample quantity is divided by the calibrator quantity, the unit from the
standard curve cancels out. Therefore, all that is required of the standards is that their
relative dilutions be known. For relative quantification, this means any stock RNA or
DNA containing the appropriate target can be used to prepare standards.

How to Perform To perform the relative standard curve method for quantification:
the Relative • Perform a run on your real-time PCR instrument, this includes:
Standard Curve
– Setting up a reaction plate
Method
– Analyzing the data

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 Relative Standard Curve Method for Quantification

– Creating a standard curve

• Determine the relative values.
See “Example of the Relative Standard Curve Method” below for an illustration of
these steps.

Example of the This example illustrates the use of standard curves for relative quantification, based
Relative Standard on:
Curve Method • The target is human c-myc mRNA, and the endogenous control is human
GAPDH mRNA.
• The target and endogenous control are amplified in separate tubes.
• Dilutions of a cDNA sample prepared from total Raji RNA are used to construct
standard curves for the c-myc and the GAPDH amplifications.
• The unknown samples (samples to characterize) are cDNA prepared from total
RNA isolated from human brain, kidney, liver, and lung.

Performing the Run

The procedure below is a general outline for performing a run. Refer to your
instrument user manual for detailed instructions.

1. Set up a reaction plate.

2. Place the reaction plate on your real-time PCR instrument and start the run.

3. When the run is complete, analyze the data.

4. Set the threshold and create a standard curve from the data.
The figure below shows the standard curve for the amplification of the c-
myc target detected using a FAM dye labeled probe.

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Chapter 3 Gene Expression and Other Quantitative Assays

Determining the Relative Values

To determine the relative value:

1. Calculate the log input amount by entering the following formula in one cell
of the work sheet of any spreadsheet program:
= ([cell containing CT value] – b)/m
where b = y-intercept of standard curve line and m = slope of standard curve
line
Note: In this example, b = 25.712 and m = –3.385
for the equation y = mx + b.

2. Calculate the input amount by entering in an adjacent cell the following

formula:
= 10^ [cell containing log input amount]
Note: The units of the calculated amount are the same as the units used to
construct the standard curve, which are nanograms of Total Raji RNA. If it is
calculated that an unknown has 0.23 ng of Total Raji RNA, then the sample
contains the same amount of c-myc mRNA found in 0.23 ng of the Raji
Control RNA.

3. Repeat the steps to construct a standard curve for the endogenous reference
using the CT values determined with the GAPDH probe. Refer to Table 3-18
on page 3-40.

4. Because c-myc and GAPDH are amplified in separate tubes, average the c-
myc and GAPDH values separately.

5. Divide the amount of c-myc by the amount of GAPDH to determine the

normalized amount of c-myc (c-mycN).

6. Designate the calibrator.

In Table 3-18 on page 3-40, brain is arbitrarily designated as the calibrator.

7. Divide the averaged sample (kidney, liver, or lung) value by the averaged
calibrator (brain) value. Calculate the coefficient of variation (see below),
based on the cv of the sample and brain.

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 Relative Standard Curve Method for Quantification

Calculating the Coefficient Of Variation

The c-mycN value is determined by dividing the average c-myc value by the average
GAPDH value. The standard deviation of the quotient is calculated from the standard
deviations of the c-myc and GAPDH values using the following formula:

2
cv = cv 1 + cv 22

where:

cv = ---s- = ----------------------------
stddev
X meanvalue

cv 1 = cv c – myc

cv 2 = cvGAPDH

As an example, from Table 3-18 on page 3-40 (brain sample):

stddev c – myc
- = 0.004
cv 1 = ------------------------------------------- -------------
meanvalue c – myc 0.039

and

stddev GAPDH
- = 0.034
cv 2 = --------------------------------------------- -------------
meanvalue GAPDH 0.54

2 2
cv = ⎛ 0.004 0.034
-------------⎞ + ⎛ -------------⎞ = 0.12
⎝ 0.039⎠ ⎝ 0.54 ⎠

since

cv = ---s-
X

meanvalue c – myc
s = ( cv ) ( X ) = ( cv ) ⎛⎝ ----------------------------------------------⎞⎠
meanvalue GAPDH
0.039
s = ( 0.12 ) ⎛ -------------⎞ = ( 0.12 ) ( 0.07 )
⎝ 0.54 ⎠

s = 0.008

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Chapter 3 Gene Expression and Other Quantitative Assays

Comparing Samples with a Calibrator

The normalized amount of target (c-mycN) is a unitless number that can be used to
compare the relative amount of target in different samples. One way to make this
comparison is to designate one of the samples as a calibrator. In Table 3-18 on
page 3-40, brain is designated as the calibrator; brain is arbitrarily chosen because it
has the lowest expression level of the target.

Relative Standard Curve Method Results

Each c-mycN value in Table 3-18 on page 3-40 is divided by the brain c-mycN value
to give the values in the final column. These results indicate the kidney sample
contains 5.5 times as much c-myc mRNA as the brain sample, liver 34.2 times as
much, and lung 15.7 times as much.
Note: Averages and standard deviations are calculated from unrounded data, not the
rounded data presented here.

Table 3-18 Amounts of c-myc and GAPDH in human brain, kidney, liver, and lung tissues

c-myc GAPDH c-mycN c-mycN

Tissue
ng Total Raji RNA ng Total Raji RNA Norm. to GAPDH a Rel. to Brain b

Brain 0.033 0.51

(Designated
calibrator) 0.043 0.56

0.036 0.59

0.043 0.53

0.039 0.51

0.040 0.52

Average 0.039 ±0.004 0.54 ±0.034 0.07 ±0.008 1.0 ±0.12

Kidney 0.40 0.96

0.41 1.06

0.41 1.05

0.39 1.07

0.42 1.06

0.43 0.96

Average 0.41 ±0.016 1.02 ±0.052 0.40 ±0.025 5.5 ±0.35

Liver 0.67 0.29

0.66 0.28

0.70 0.28

0.76 0.29

0.70 0.26

0.68 0.27

Average 0.70 ±0.036 0.28 ±0.013 2.49 ±0.173 34.2 ±2.37

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 Relative Standard Curve Method for Quantification

Table 3-18 Amounts of c-myc and GAPDH in human brain, kidney, liver, and lung tissues (continued)

c-myc GAPDH c-mycN c-mycN

Tissue
ng Total Raji RNA ng Total Raji RNA Norm. to GAPDH a Rel. to Brain b

Lung 0.97 0.82

0.92 0.88

0.86 0.78

0.89 0.77

0.94 0.79

0.97 0.80

Average 0.93 ±0.044 0.81 ±0.041 1.15 ±0.079 15.7 ±1.09

a. The c-mycN value is determined by dividing the average c-myc value by the average GADPH value. The standard
deviation of the quotient is calculated from the standard deviations of the c-myc and GADPH values.
b. The calculation of c-mycN relative to brain involves division by the calibrator value. This is a division by an arbitrary
constant, so the cv of this result is the same as the cv for c-mycN.

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Chapter 3 Gene Expression and Other Quantitative Assays

Comparative CT Method for Relative Quantification

The comparative CT method is similar to the relative standard curve method, except
that it uses an arithmetic formula rather than a standard curve to achieve the same
result for relative quantification.
When using the comparative CT method with real-time PCR, you can normalize to an
endogenous reference using data generated during the PCR experiment. This is
especially useful when you have a limited amount of RNA or when you perform
high-throughput processing of a large number of samples (Livak and Schmittgen,
2001).
IMPORTANT! You can eliminate the use of standard curves for relative
quantification if you perform a validation experiment. (See “Performing the
Validation Experiment” below.)

Formula The amount of target, normalized to an endogenous control and relative to a

calibrator, is calculated using:
2 –∆∆CT
See Appendix A, “Formulas,” for a derivation of the formula.

Performing the Validation Experiment

Before using the ∆∆CT method for quantification, perform a validation experiment
like the one below to verify that efficiencies of target and reference are
approximately equal.

To perform the validation experiment:

1. Choose your target and endogenous control.

2. Perform a dilution series of different input amounts for your target and
endogenous control.
Note: When possible, serial dilutions should cover 5 to 6 orders of magnitude.

3. Run the samples on your real-time PCR instrument.

4. Analyze your data.

5. Calculate the average CT and ∆CT values for your target and endogenous
control (see Table 3-19 on page 3-44).

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 Comparative CT Method for Relative Quantification

To perform the validation experiment: (continued)

6. Plot the log of the input amount vs. ∆CT as explained on page 3-38 (see the
figure below).

As a guideline, the absolute value of the slope of log input amount vs. ∆CT
should be less than 0.1.
The slope in the figure above is –0.0034, which passes this test.
Note: The less-than-0.1 guideline should provide gene expression results with a
low degree of experimental variation. However, if you must quantify any variation
introduced by the comparative CT method, you need to perform and compare the
results of parallel experiments using the comparative CT and the standard curve
method.

If the efficiencies of the two

Then...
systems are...

< 0.1 • You can use the ∆∆CT calculation for the
relative quantification of target without
running standard curves on the same plate.

> 0.1 • Perform the validation experiment over a

larger dynamic range (5 to 6 orders of
magnitude) or design and synthesize new
primers to improve efficiency.
• Perform the quantification using the
standard curve method.

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Chapter 3 Gene Expression and Other Quantitative Assays

Table 3-19 Average CT value for c-myc and GAPDH at different input amounts

Input Amount c-myc GAPDH ∆CT

ng Total RNA Average CT Average CT c-myc – GAPDH

100.0 20.28 +0.11 15.23 +0.02 5.05 +0.11

10.0 23.88 +0.14 18.62 +0.04 5.26 +0.15

1.0 27.33 +0.09 22.18 +0.02 5.15 +0.09

0.1 30.93 +0.23 25.53 +0.07 5.40 +0.24

0.01 34.39 +0.37 29.13 +0.14 5.26 +0.39

0.001 37.03 +0.12 32.57 +0.13 4.46 +0.17

Relative For the ∆∆CT calculation to be valid, the efficiency of the target amplification and
Efficiencies of the efficiency of the reference amplification must be approximately equal. To assess
Target and if two amplicons have the same efficiency, you can look at how ∆CT varies with
Reference template dilution. The standard curves for c-myc and GAPDH used in the previous
section provide the necessary data. Table 3-19 shows the average CT value for c-myc
and GAPDH at different input amounts.

How to Perform To perform the comparative CT method for relative quantification:

the Comparative • Perform a run on your real-time PCR instrument. This includes:
CT Method
– Setting up a reaction plate
– Analyzing the data
• Determine the ∆CT value (Target − Endogenous control).
• Calculate the ∆∆CT to determine fold difference in gene expression
(∆CT Target − ∆CT Calibrator).
See “Example of the Comparative CT Method” below.

Example of the This example illustrates the use of the comparative CT method for relative
Comparative CT quantification. In this example:
Method • The target is human c-myc mRNA and the endogenous control is human
GAPDH mRNA.
• The target and endogenous control are amplified in separate tubes.
• The unknown samples are cDNA prepared from total RNA isolated from human
brain, kidney, liver, and lung.

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 Comparative CT Method for Relative Quantification

Performing the Run

The procedure below is a general outline for performing a run. Refer to your
instrument user manual for detailed instructions.

To perform the run:

1. Set up a reaction plate.

2. Place the reaction plate on your instrument and start the run.

3. When the run is complete, analyze the data.

Comparative CT Method Results

The CT data used to determine the amounts of c-myc and GAPDH mRNA shown in
Table 3-19 on page 3-44 are used to illustrate the ∆∆CT calculation. Table 3-20 on
page 3-46 shows the average CT results for the human brain, kidney, liver, and lung
samples and how these CT s are manipulated to determine ∆CT, ∆∆CT, and the relative
amount of c-myc mRNA. The results are comparable to the relative c-myc levels
determined using the standard curve method as shown in Table 3-18.
Although the comparative CT method can be used to make this type of tissue
comparison, biological interpretation of the results is complex. The single relative
quantity reported actually reflects variation in both target and reference transcripts
across a variety of cell types that might be present in any particular tissue (Livak and
Schmittgen, 2001).

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Chapter 3 Gene Expression and Other Quantitative Assays

Table 3-20 Relative quantification using the comparative CT method

Average CT RQ e
Average ∆CT
∆CT −∆∆CT RQ c-mycN
Tissue Endogenous c-myc–
Target VAB b −(∆CT–∆CT, Brain) c Rel. to Brain d
Control GAPDH a Min Max
c-myc
GAPDH

Brain 30.49 23.63 6.86 0.087 0.00 1.0 0.9 1.2

Calibrator

Kidney 27.03 22.66 4.37 0.050 2.50 5.6 5.2 6.2

Liver 26.25 24.60 1.65 0.049 5.21 37.0 34.0 40.3

Lung 25.83 23.01 2.81 0.049 4.05 16.5 15.2 18.0

a. The average ∆CT value is determined by subtracting the average GAPDH CT value from the average c-myc CT value.
For example, ∆CT Brain = 30.49 – 23.63 = 6.86.
b. VAB = Applied Biosystems’ variability function for calculating the variability of the test sample ∆CT statistic.
c. The calculation of –∆∆CT involves subtracting ∆CT calibrator value from the ∆CT target value.
For example, –∆∆CT Kidney = –(∆CT Kidney –∆CT Brain) = –(4.37 – 6.86) = 2.50 (using full data value, not the rounded data
presented in this table).
d. The RQ for c-myc relative to brain is calculated using the equation: 2 –(∆∆CT).
For example, the Kidney sample has a ∆∆CT value of –2.50. Therefore, 2 –(∆∆CT) = 2-(-2.50) = 5.7.
e. The RQ minimum and RQ maximum define statistical boundaries for relative quantification, based upon a user-specified
RQ Min/Max confidence setting. For example, a confidence setting of 95.00% means that the user can expect the true
RQ value to fall within the RQ Min/Max range with a 95% confidence. The RQ Min/Max is calculated using the equation:
2–(∆∆CT(s,t)±T × VAB(CT(s,t))), where ∆∆CT(s,t) = ∆CT(s,t) - ∆CT(calibrator,t), s = sample name, t = target detector, T = student’s T value
computed at the selected confidence setting using a degrees of freedom that is associated with the test sample ∆CT(s,t),
and VAB is as defined in footnote b above.
Note: These example experiment results are from a single plate study using four replicates per target and endogenous
controls for each tissue.

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 Multiplex PCR (Same-Tube) Method for Relative Quantification

Multiplex PCR (Same-Tube) Method for Relative

Quantification
Multiplex PCR is the use of more than one primer pair and probe in the same tube.
You can use this method in relative quantification where one primer pair/probe
amplifies the target and another primer pair/probe amplifies the endogenous control
in the same tube.
You can perform a multiplex reaction for both the standard curve method and the
comparative CT method.

Advantages of The advantages of performing target and reference reactions in the same tube are:
Multiplex PCR • Higher throughput is most evident if you are interested in analyzing a single
target because the number of sample tubes is reduced by a factor of two.
• More efficient use of samples
• Reduction in reagent use and cost
See “Primer Limiting in Multiplex Assays” on page 3-28.

About Multiple The TaqMan® Probe-based chemistry includes multiple reporter dyes, which make it
Reporter Dyes possible to amplify and detect target amplicon and endogenous control amplicon in
the same tube.
The reporter dyes recommended for TaqMan probes are FAM™ and VIC® dyes.
These dyes are distinguishable from one another because they have different
emission wavelength maxima:
• FAM dye: λmax = 518 nm
• VIC dye: λmax = 554 nm

About Multi- The software for all of the real-time PCR instruments uses a process called
componenting multicomponenting to distinguish reporter dyes, the quencher dye TAMRA™ (λmax =
582 nm), if used, and the passive reference ROX™ dye (λmax = 610 nm).
Multicomponenting is a mathematical algorithm that uses pure dye reference spectra
to calculate the contribution of each dye to a complex experimental spectrum. When
using TaqMan MGB probes, no quencher dye (TAMRA) is necessary. Because there
is one less dye to resolve, spectral resolution is improved.

Obtaining Accurate Quantification

For the most accurate quantification using two probes in one tube, use the reporter
dyes that have the largest difference in emission maximum: FAM and VIC dye.

About Primer Reactions designed to amplify two different sequences in the same tube share
Limitation common reagents. If the two sequences have different initial copy numbers, it is
possible for the more abundant species to use up these common reagents, impairing
amplification of the rarer species. For accurate quantification, it is important that the
two reactions do not compete. Competition can be avoided by limiting the
concentration of primers used in the amplification reactions. For more information
on primer limitations, see page 3-28.

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Chapter 3 Gene Expression and Other Quantitative Assays

How to Perform To perform the multiplex PCR with the relative standard curve method for
Multiplex PCR quantification:
with the Relative • Perform a run on your real-time PCR instrument. This includes:
Standard Curve
– Setting up a reaction plate
Method
– Analyzing the data
– Creating a standard curve
• Determine the relative values.
See the example below for an illustration of these steps.

Example of This example illustrates the use of multiplex PCR with the relative standard curve
Multiplex PCR method for quantification. In this example:
with the Relative • The target is human c-myc mRNA and the endogenous control is human
Standard Curve GAPDH mRNA.
Method • The target and endogenous control are amplified in the same tube.
• Dilutions of a cDNA sample prepared from Total Raji RNA are used to
construct standard curves for the c-myc and the GAPDH amplifications.
• The unknown samples are cDNA prepared from total RNA isolated from human
brain, kidney, liver, and lung.

Performing the Run

The same-tube and separate tube procedures for the relative standard curve method
are identical, with the exceptions noted below.

To perform the run:

1. Perform the run per the procedures on page 3-37.

Exception: When you set up the reaction plate, the target amplicon and
endogenous control amplicon are in the same tube.
Exception: ∆CT is calculated for each reaction and then the ∆CTs are
averaged.

2. Determine the relative values per the procedures in Table 3-17 on page 3-38.

Results of the Above Run

Table 3-21 shows the results of a same-tube experiment using the relative standard
curve method. In this experiment, the target c-myc normalized to the endogenous
control GAPDH was quantified.
Averages and deviations are calculated from unrounded data, not the rounded data
presented here.

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 Multiplex PCR (Same-Tube) Method for Relative Quantification

Table 3-21 Relative quantification using multiplex PCR with the relative standard curve method

c-myc GAPDH c-mycN c-mycN

Tissue
ng Total Raji RNA ng Total Raji RNA Norm. to GAPDH Rel. to Brain

Brain 0.031 0.618 0.05

0.038 0.532 0.07

0.032 0.521 0.06

0.038 0.550 0.07

0.032 0.577 0.06

0.037 0.532 0.07

Average 0.06 ±0.008 1.0 ±0.14

Kidney 0.365 0.049 0.35

0.338 1.035 0.33

0.423 1.042 0.41

0.334 1.086 0.31

0.334 1.021 0.33

0.372 1.139 0.33

Average 0.34 ±0.035 5.4 ±0.55

Liver 0.477 0.255 1.87

0.471 0.228 2.06

0.535 0.258 2.07

0.589 0.241 2.44

0.539 0.264 2.04

0.465 0.227 2.05

Average 2.09 ±0.186 33.3 ±2.97

Lung 0.853 0.085 0.97

0.900 0.084 0.88

0.956 0.082 1.00

0.900 0.093 0.87

0.996 0.112 0.87

0.859 0.090 0.84

Average 0.90 ±0.062 14.4 ±0.99

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Chapter 3 Gene Expression and Other Quantitative Assays

How to Perform To perform multiplex PCR with the comparative CT method for relative
Multiplex PCR quantification:
with the • Perform a validation experiment.
Comparative CT
• Perform a run on your real-time PCR instrument. This includes:
Method
– Setting up a reaction plate
– Analyzing the data
• Determine the ∆CT value.
• Perform the ∆∆CT calculation.
See the example below for an illustration of these steps.

Example of This example illustrates the use of multiplex PCR with the comparative CT method
Multiplex PCR for relative quantification. In this example:
with the • The target is human c-myc mRNA and the endogenous control is human
Comparative CT GAPDH mRNA.
Method • The target and endogenous control are amplified in the same tubes.
• The unknown samples are cDNA prepared from total RNA isolated from human
brain, kidney, liver, and lung.

Procedures
The same-tube and separate tube procedures for the comparative CT method are
identical, with the exceptions noted below.

To perform the run:

1. Perform the validation experiment per the procedures on page 3-42.

2. Perform the run per the procedures on page 3-45.

Exception: When you set up the reaction plate, the target amplicon and
endogenous control amplicon are in the same tube.
Exception: Because c-myc and GAPDH data are being obtained from the
same tube, calculations are carried out individually for each well before
averaging.

3. Determine the ∆CT value per the procedures on page 3-44.

4. Perform the ∆∆CT calculation per the procedures on page 3-44.

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 Multiplex PCR (Same-Tube) Method for Relative Quantification

Results of the Run

Table 3-22 shows the ∆∆CT calculations for the same-tube experiment.

Table 3-22 Relative quantification using multiplex PCR with the comparative CT method

∆∆CT
∆CT c-mycN
Tissue c-myc CT GAPDH CT ∆CT – Avg. ∆CT,
c-myc – GAPDH Rel. to Brain
Brain

Brain 32.38 25.07 7.31

32.08 25.29 6.79

32.35 25.32 7.03

32.08 25.24 6.84

32.34 25.17 7.17

32.13 25.29 6.84

Average 6.93 ±0.16 0.00 ±0.16 1.0

(0.9 to 1.1)

Kidney 28.73 24.30 4.43

28.84 24.32 4.52

28.51 24.31 4.20

28.86 24.25 4.61

28.86 24.34 4.52

28.70 24.18 4.52

Average 4.47 ±0.14 –2.47 ±0.14 5.5

(5.0 to 6.1)

Liver 28.33 26.36 1.97

28.35 26.52 1.83

28.16 26.34 1.82

28.02 26.44 1.58

28.15 26.31 1.84

28.37 26.53 1.84

Average 1.81 ±0.13 –5.12 ±0.13 34.8

(31.9 to 38.0)

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Chapter 3 Gene Expression and Other Quantitative Assays

Table 3-22 Relative quantification using multiplex PCR with the comparative CT method (continued)

∆∆CT
∆CT c-mycN
Tissue c-myc CT GAPDH CT ∆CT – Avg. ∆CT,
c-myc – GAPDH Rel. to Brain
Brain

Lung 27.47 24.55 2.92

27.39 24.33 3.06

27.30 24.43 2.87

27.39 24.32 3.07

27.24 24.18 3.06

27.46 24.34 3.12

Average 3.02 ±0.10 –3.92 ±0.10 15.1

(14.1 to 16.2)

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 Standard Curve Method for Absolute Quantification

Standard Curve Method for Absolute Quantification

Unlike the standard curve method for relative quantification, the standard curve
method for absolute quantification depends on knowing the absolute quantities of the
standards.

Requirements Quantification by the absolute standard curve method requires that:

• The DNA or RNA be a single, pure species. For example, plasmid DNA
prepared from E. coli often is contaminated with RNA, which increases the A260
measurement and inflates the copy number determined for the plasmid.
• You pipette accurately because the standards must be diluted over several orders
of magnitude. Plasmid DNA or in vitro transcribed RNA must be concentrated
to measure an accurate A260 value. This concentrated DNA or RNA must then
be diluted 106 to 1012 -fold to be at a concentration similar to the target in
biological samples.
• The stability of the diluted standards be considered, especially for RNA. Divide
diluted standards into small aliquots, store at –80 °C, and thaw only once before
use. An example of the effort required to generate trustworthy standards is
provided by Collins (Collins et al. 1995), who report on the steps they used to
develop an absolute RNA standard for viral RNA quantification.
• You not use DNA as a standard for absolute quantification of RNA because
there is no control for the efficiency of the reverse transcription step.

Standards Plasmid DNA and in vitro transcribed RNA are commonly used to prepare absolute
standards. Concentration is measured by A260 and converted to the number of copies
using the molecular weight of the DNA or RNA.

How to Perform Except for preparation of the standards (see above), the absolute standard curve
the Absolute methods and relative standard curve quantification methods are identical.
Standard Curve To perform the absolute standard curve method for quantification, see:
Method
• “How to Perform the Relative Standard Curve Method” on page 3-36
• “Example of the Relative Standard Curve Method” on page 3-37

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Chapter 3 Gene Expression and Other Quantitative Assays

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Allelic Discrimination Assays 4 4
This chapter covers:
About Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
Purchasing an Applied Biosystems TaqMan SNP Genotyping Assay Product. . . . 4-4
TaqMan Pre-Developed Assay Reagents for Allelic Discrimination . . . . . . . . . . . 4-6
Designing Your Own Allelic Discrimination Assay . . . . . . . . . . . . . . . . . . . . . . . . 4-6

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Chapter 4 Allelic Discrimination Assays

About Allelic Discrimination Assays

What Is an Allelic An Allelic Discrimination Assay is an endpoint assay used to determine the genotype
Discrimination of samples. With this assay type, you can differentiate a single nucleotide
Assay? polymorphism (SNP).
An Allelic Discrimination Assay determines if unknown samples are:
• Homozygotes (samples having only allele 1)
• Homozygotes (samples having only allele 2)
• Heterozygotes (samples having both allele 1 and allele 2)

Instruments Allelic discrimination assays can be used with the following instruments:
• Applied Biosystems 7900HT Fast Real-Time PCR System (7900HT System)
• ABI PRISM® 7900HT Sequence Detection System, upgradeable to the Applied
Biosystems 7900HT Fast Real-Time PCR System with the 7900HT System Fast
Service Upgrade
• Applied Biosystems 7300 Real-Time PCR System (7300 System)
• Applied Biosystems 7500 Real-Time PCR System (7500 System), upgradeable
to the Applied Biosystems 7500 Fast Real-Time PCR System with the 7500 Fast
Real-Time PCR Upgrade Kit
• Applied Biosystems 7500 Fast Real-Time PCR System (7500 Fast System)
Note: You can run allelic discrimination assays on a Fast-capable 7900HT System or
7500 Fast System using standard reagents; Allelic Discrimination Assays are not
supported using Fast reagents and protocols.
Note: Use of the 7900HT, 7500 Fast, 7500, or 7300 Systems allows for Real-Time
analysis of PCR, which is helpful for troubleshooting. If using a real-time PCR
system for PCR amplification, perform the endpoint plate read separately.

Chemistry Allelic Discrimination Assays can be used with fluorogenic 5′ nuclease chemistry
(also known as TaqMan® Probe-based chemistry. For information, see “TaqMan
Probe-Based Chemistry” on page 2-3).
Note: The SYBR® Green I dye chemistry and the TaqMan® Fast Universal PCR
Master Mix (2✕), No AmpErase® UNG, are not supported for Allelic Discrimination
Assays.

Terms Used
in Allelic Table 4-1 Terms used in allelic discrimination analysis
Discrimination
Analysis Term Definition

No template control A sample that does not contain template. The NTC shows
(NTC) background signal and is used as the negative control. It
provides a means of measuring contamination that might give
a false positive signal.

Nucleic acid target Nucleotide sequence that you want to detect.

(also called “target
template”)

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 About Allelic Discrimination Assays

Table 4-1 Terms used in allelic discrimination analysis (continued)

Term Definition

Passive reference A dye that provides an internal reference to which the

reporter dye signal is normalized during data analysis.
Normalization is necessary to correct for fluorescence
fluctuations caused by changes in concentration or volume. A
passive reference dye is included in all Applied Biosystems
real-time PCR reagent kits.

Reporter dye The dye attached to the 5′ end of a TaqMan probe.

Normalized reporter The normalized intensity of the reporter dye.

(Rn )
Rn is calculated by dividing the intensity of the reporter dye by
the intensity of the passive reference.

Unknown The sample you want to classify as either homozygote or

heterozygote.

How Allelic In allelic discrimination assays, the PCR includes a specific, fluorescent, dye-labeled
Discrimination probe for each allele. You can use TAMRA™ dye or TaqMan MGB labeled probes.
Assays Work The probes contain different fluorescent reporter dyes (FAM™ dye and VIC® dye) to
differentiate the amplification of each allele.
Each TaqMan MGB probe contains:
• A reporter dye at the 5′ end of each probe
– VIC dye is linked to the 5′ end of the Allele 1 probe
– FAM dye is linked to the 5′ end of the Allele 2 probe
• A minor groove-binder (MGB)
This modification increases the melting temperature (Tm) without increasing
probe length (Afonina et al., 1997; Kutyavin et al., 1997), thereby allowing the
design of shorter probes. This type of probe design results in greater differences
in Tm values between matched and mismatched probes, which produces more
accurate allelic discrimination.
• A nonfluorescent quencher (NFQ) at the 3′ end of the probe
Because the quencher does not fluoresce, real-time PCR systems can measure
reporter dye contributions more accurately.
During PCR, each probe anneals specifically to complementary sequences between
the forward and reverse primer sites. AmpliTaq Gold ® DNA polymerase can cleave
only probes that hybridize to the allele sequence. Cleavage separates the reporter dye
from the quencher dye, which results in increased fluorescence by the reporter dye.
Thus, the fluorescence signal(s) generated by PCR amplification indicate(s) the
alleles that are present in the sample.

Mismatches Between Probe and Allele Sequences

Mismatches between a probe and allele (Figure 4-1) reduce the efficiency of probe
hybridization. Furthermore, AmpliTaq Gold DNA polymerase is more likely to
displace the mismatched probe rather than cleave it to release reporter dye.

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Chapter 4 Allelic Discrimination Assays

Allele F
1 V Legend
Q Q
V VIC dye

Match Mismatch
F FAM dye

Q Quencher
Allele F V
2
Q Q AmpliTaq
Gold DNA
Polymerase

Match Mismatch GR1556

Figure 4-1 Results from matches and mismatches between allele and probe
sequences in allelic discrimination assays

Table 4-2 summarizes the possible results of the allelic discrimination assays
example shown above.

Table 4-2 Allelic discrimination assay results

A substantial increase in… Indicates…

VIC dye fluorescence only homozygosity for Allele 1.

FAM dye fluorescence only homozygosity for Allele 2.

both fluorescent signals heterozygosity.

Purchasing an Applied Biosystems TaqMan SNP

Genotyping Assay Product

TaqMan SNP Genotyping Assays

Product TaqMan® SNP Genotyping Assays provide the largest collection of ready-to-use
Description SNP assays available for human studies. All assays are designed using Applied
Biosystems bioinformatics pipeline and software, as well as genomic information
from Celera Genomics and public databases:
• TaqMan® Validated SNP Genotyping Assays: SNP genotyping assays which
have undergone minor allele frequency (MAF) validation on two to four ethnic
group populations (45 individual samples per ethnic group) and inventoried for
fast availability.
• TaqMan® Coding SNP Genotyping Assays: SNP genotyping assays for the
detection of informative and putative functional SNPs in gene-coding regions.
These inventoried assays are functionally tested to assure quality performance.

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 Purchasing an Applied Biosystems TaqMan SNP Genotyping Assay Product

• TaqMan® Pre-Designed SNP Genotyping Assays: Genome-wide and genome-

unique in silico SNP genotyping assays. Manufactured and functionally tested
upon ordering.
TaqMan SNP Genotyping Assays provide optimized assays for genotyping single
nucleotide polymorphisms (SNPs). The products use the 5′ nuclease assay for
amplifying and detecting specific SNP alleles in purified genomic DNA samples.
Each assay allows researchers to genotype individuals for a specific SNP.

Available To view the available TaqMan SNP Genotyping Assays (PN 4331183 and 4351379)
Products go to:
http://www.appliedbiosystems.com
For information about ordering TaqMan SNP Genotyping Assays, see “How to
Obtain Support” on page ix.

Product TaqMan SNP Genotyping Assays:

Properties • Are designed and optimized to work with the TaqMan® 2✕ Universal PCR
Master Mix, No AmpErase® UNG, using the same universal thermal cycling
conditions. This protocol design facilitates the workflow for all throughput
requirements and in any SNP genotyping study.
• The assays require only three components:
– 1 to 20 ng of purified genomic DNA sample
– 20✕, 40✕, or 80✕ SNP Genotyping Assay Mix (specific for each
polymorphism)
– TaqMan 2✕ Universal PCR Master Mix (with or without AmpErase UNG)
• Require only one amplification step and an endpoint reading to obtain results.

Assay Contents Each TaqMan SNP Genotyping Assay consists of:

• One tube containing 20✕, 40✕, or 80✕ SNP Genotyping Assay Mix.
Concentration depends on product and scale ordered.
• CD-ROM containing assay information and PDFs of the protocol and product
insert

About SNP The SNP Genotyping Assay Mix contains:

Genotyping • Sequence-specific forward and reverse primers to amplify the SNP of interest
Assay Mix
• Two TaqMan MGB probes:
– One probe labeled with VIC dye detects the Allele 1 sequence
– One probe labeled with FAM dye detects the Allele 2 sequence

About the Assay The assay information file consists of:

Information File • Genomic information about the SNP, including the chromosomal location, allele
(AIF) frequency (for validated assays), and context sequence
• Information about the packaging of each assay tube, including the location in
the plate rack and the 2-D bar code

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Chapter 4 Allelic Discrimination Assays

Custom TaqMan SNP Genotyping Assays Service

Product If a particular SNP of interest is not available as a product on the Applied Biosystems
Description Web site, you can use our Custom TaqMan® SNP Genotyping Assay Service to
submit sequences. See “Custom TaqMan SNP Genotyping and Gene Expression
Assays” on page 1-7 for additional details on Custom TaqMan® Assays. More
information about Custom TaqMan SNP Genotyping Assays and related products,
part numbers, and free software is also available at:
http://www.allsnps.com
To place an order, contact your Applied Biosystems representative.

Product Each Custom TaqMan® SNP Genotyping Assay is manufactured based solely on the
Properties customer’s input target sequence. All information supplied by the customer and
returned by Applied Biosystems to the customer is considered confidential and is
treated accordingly. Visit the Applied Biosystems support web site to access tutorials
on how to submit sequences to the Custom TaqMan® Gene Expression Assay
Service. See “How to Obtain Support” on page ix.

TaqMan Pre-Developed Assay Reagents for Allelic

Discrimination
Product TaqMan® Pre-Developed Assay Reagents for Allelic Discrimination (TaqMan®
Description PDARs for Allelic Discrimination) are optimized assays for the discrimination of
specific alleles.
TaqMan PDARs for Allelic Discrimination require only three components:
• Genomic DNA sample
• 10✕ Allelic Discrimination Assay Mix (specific for each polymorphism)
• 2✕ TaqMan® Universal PCR Master Mix
Note: Allele 1 and 2 control DNA is included with each assay, allowing each
homozygote signal to be generated on each run.
For instructions on how to use TaqMan PDARs for Allelic Discrimination, refer to
the Pre-Developed TaqMan® Assay Reagents Allelic Discrimination Protocol
(PN 4312214).

Designing Your Own Allelic Discrimination Assay

Design and When you design your own allelic discrimination assays:
Optimization • Design probes and primers using Primer Express® software
Steps
• Select the appropriate reagent configuration
• Use universal thermal cycling parameters

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 Designing Your Own Allelic Discrimination Assay

• Use default primer and probe concentrations

IMPORTANT! These steps provide a rapid and reliable system for assay design and
optimization only when used in their entirety. The system must be adopted as a whole
in order to achieve the highest level of success, due to the interdependence of many
of the individual components.

Conclusions From the results of thousands of assays run according to these guidelines, the
following conclusion can be made:
You can use 900-nM primers, a 200-nM probe, and 1 to 20 ng of genomic DNA
to achieve reproducible and sensitive assay results.

Probe Design Using Primer Express Software

The Primer Express software uses a set of default parameters to automatically select
primer and probe sets.
The probe design guidelines for allelic discrimination assays are discussed here.
After choosing probes based on the guidelines below, you design the primer. For
detailed information, see the Primer Express Software Version 3.0 Getting Started
Guide (PN 4362460).

TaqMan MGB The TaqMan MGB probes are two modified conventional TaqMan probes:
Probes • One probe matches the Allele 1 sequence
• One probe matches the Allele 2 sequence
Table 4-3 below summarizes the TaqMan MGB probe features.

Table 4-3 Taqman MGB probe features

Probe 5′ Label 3′ Label Other Features

TaqMan MGB FAM or VIC Nonfluorescent minor groove-binder

dye quencher

When to Use Applied Biosystems recommends the general use of TaqMan MGB probes for allelic
TaqMan MGB discrimination assays:
Probes • To achieve estimated Tm values of 65 to 67 °C using probes shorter than
25 nucleotides.
• To obtain greater differences in Tm values between matched and mismatched
probes than with conventional TaqMan probes.
• To obtain more precise measurements of dye contributions.

Allelic Discrimina- IMPORTANT! When designing probes, it is important to consider probes from both
tion Probe Design strands.
Guidelines • To label allelic discrimination probes, use VIC or FAM dyes.
• Avoid probes with G residue at the 5′ end of the probe. A G residue adjacent to
the reporter dye will quench the reporter fluorescence, even after cleavage.
• Select probes using Primer Express software (estimated Tm of 65 to 67 °C).

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Chapter 4 Allelic Discrimination Assays

• Make TaqMan MGB probes as short as possible without being shorter than
13 nucleotides.
• Avoid runs of an identical nucleotide. This guideline is especially true for
guanine, where runs of four or more Gs should be avoided.
• Position the polymorphic site in the central third of the probe.
Note: The polymorphic site can be shifted toward the 3′ end to meet the above
guidelines, however, the site must be located more than two nucleotides upstream
from the 3′ terminus.
Figure 4-2 illustrates the placement of a polymorphism in an example probe
(N = Nucleotide).

Polymorphism
If necessary, place the
polymorphism here

5′ 3′
N N N N N N N N N N N N N N N N N N N N N

First try to position the polymorphic Do not place the

site in the central third of the probe polymorphism here

Figure 4-2 Polymorphism placement in an example probe

Primer Design Using Primer Express Software

The Primer Express software uses a set of default parameters to automatically select
primer and probe sets.
The primer design guidelines for allelic discrimination assays are discussed here. For
more information, refer to the Primer Express Software Version 3.0 Getting Started
Guide (PN 4362460). After selecting probes for the assay (see page 4-7), choose
primers based on the guidelines below.
Note: Primer Express software is designed for universal assay conditions. Changing
assay conditions or Master Mix may result in suboptimal performance.

Primer Design If you follow the guidelines below, the amplicons should be 50 to 150 basepairs. By
Guidelines limiting the parameters for amplicon design (such as amplicon size), it is possible to
run all reactions with a single reaction buffer (such as TaqMan 2✕ Universal PCR
Master Mix) and a single thermal cycling protocol.
• Avoid runs of an identical nucleotide. This guideline is especially true for
guanine, where runs of four or more should be avoided.
• The Tm of the primers should be 58 to 60 °C.
• Keep the G/C content within 30 to 80%.
• Make sure the last five nucleotides at the 3′ end contain no more than two G/C
residues.
• Place the forward and reverse primers as close as possible to the probe without
overlapping it.

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 Designing Your Own Allelic Discrimination Assay

Selecting the Appropriate Reagent Configuration

There are several TaqMan reagent kits available for allelic discrimination assays. The
reagent configuration you use depends on your particular assay.

Assays The reagent configurations listed in Table 4-4 are recommended for allelic
Containing discrimination assays. These contain the TaqMan MGB probes.
TaqMan MGB
Table 4-4 Reagent configurations for allelic discrimination assays
Probes
Product Reagent Configuration Part Number

TaqMan Reagents TaqMan Pre-Developed Go to our web site for part

Assay Reagents for Allelic numbers (Keyword: PDAR). See
Discrimination “How to Obtain Support” on
page ix.

TaqMan® 2✕ Universal PCR 4324018

Master Mix, No
AmpErase® UNG

TaqMan® SNP TaqMan® Validated and 4331183

Genotyping Assays Coding SNP Genotyping
Assays

TaqMan Pre-Designed SNP 4351379

Genotyping Assays

Custom TaqMan® Custom TaqMan® SNP Go to our web site for part
SNP Genotyping Genotyping Assays (Human) numbers (Keyword: genomic
Assays assays). See “How to Obtain
Custom TaqMan® SNP Support” on page ix.
Genotyping Assays (Non-
Human)

Quantify the amount of genomic DNA in samples before using TaqMan SNP
Genotyping Assays. Generate a standard curve using the DNA Kit (PN 401970) and
the RNase P gene primers and probe provided in the TaqMan RNase P Detection
Reagents Kit (PN 4316831).
Note: The TaqMan RNase P Detection Reagents Kit uses a TaqMan probe with
TAMRA dye as the quencher.

Using the Universal Thermal Cycling Parameters

Allelic discrimination assays designed using Applied Biosystems Assay Design
Guidelines can be run using the same universal thermal cycling parameters. This
protocol design eliminates any optimization of the thermal cycling parameters and
means that multiple assays can be run on the same plate without sacrificing
performance.

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Chapter 4 Allelic Discrimination Assays

Thermal Cycling
Parameters Table 4-5 Thermal cycling parameters for allelic discrimination assays using
TaqMan 2✕ Universal PCR Master Mix

Times and Temperatures

Initial Step PCR (Each of 40 Cycles)

AmpliTaq Gold
AmpErase UNG DNA Polymerase
Activation Melt Anneal/Extend
Activation a

HOLD CYCLE

2 min @ 50 °C 10 min @ 95 °C 15 sec @ 92 °C 1 min @ 60 °C

a. Required only if you are using TaqMan 2✕ Universal PCR Master Mix with AmpErase UNG

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Plus/Minus Assays 5 5
This chapter covers:
About Plus/Minus Assays Using an IPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2
Purchasing the Applied Biosystems TaqMan Exogenous IPC Reagents Kit . . . . . 5-4

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Chapter 5 Plus/Minus Assays

About Plus/Minus Assays Using an IPC

What Is a A plus/minus assay is an endpoint assay that indicates the presence or absence of a
Plus/Minus specific target sequence in a sample. The actual amount of target is not determined.
Assay?
Example
A plus/minus assay might be used to determine if the bacteria Salmonella is present
in hamburger meat. The results will show if Salmonella is or is not present; the
amount of bacteria is not determined.

What Is an IPC? An internal positive control (IPC) is used in plus/minus assays to monitor the PCR.
Plus/minus assays can be accomplished without an IPC, however, the IPC ensures
that a failed PCR is not mistaken for a negative test result.
The IPC consists of a template and a probe that is added to each well of a reaction
plate. Applied Biosystems has developed the TaqMan® Exogenous Internal Positive
Control Reagents kit for use in plus/minus assays. These reagents, in conjunction
with your target, identify samples that are positive or negative for a specific target
sequence. The kit distinguishes between two types of negative reactions:
• Samples identified as negative because they lack the target sequence
• Samples identified as negative because of the presence of a PCR inhibitor
Note: For more detailed information, see “Purchasing the Applied Biosystems
TaqMan Exogenous IPC Reagents Kit” on page 5-4.

Instruments Plus/minus assays using an IPC can be used with the following instruments:
• Applied Biosystems 7300 Real-Time PCR System (7300 System)
• Applied Biosystems 7500 Real-Time PCR System (7500 System)
• Applied Biosystems 7500 Fast Real-Time PCR System (7500 Fast System)
running in Standard mode
Note: The Applied Biosystems 7900HT Fast Real-Time PCR System cannot be used
for plus/minus assays using an IPC.
Note: You can run plus/minus assays on a 7500 Fast System using standard reagents;
Plus/minus assays using an IPC are not supported using Fast reagents and protocols.
These instruments are used to measure the increase of reporter fluorescence
following PCR. Reporter signals are normalized to the emission of a passive
reference, as follows:
Rn (TT) = Emission Intensity of Target Template Sequence
Emission Intensity of Passive Reference

Rn (IPC) = Emission Intensity of Internal Positive Control

Emission Intensity of Passive Reference

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 About Plus/Minus Assays Using an IPC

Chemistry Plus/minus assays using an IPC can be used with fluorogenic 5′ nuclease chemistry
(also known as TaqMan reagent or probe-based chemistry). For information, see
“TaqMan Probe-Based Chemistry” on page 2-3.
Note: The SYBR® Green I dye chemistry and TaqMan® Fast Universal PCR Master
Mix (2✕), No AmpErase® UNG, are not supported for plus/minus assays using an
IPC.

Terms Used in
Plus/Minus Table 5-1 Terms Used in Plus/Minus Analysis
Analysis
Term Definition

Internal positive control A second TaqMan probe and primer set added to the plate to
(IPC) identify well failure to amplify. Provides a means of determining
amplification failure that might give a false negative signal.

No amplification control A sample containing no target template and a blocked IPC (the
(NAC) IPC target template has been blocked by a blocking agent).

No template control A sample that does not contain template. Background signal is
(NTC) used as the negative control. Provides a means of measuring
contamination that might give a false positive signal.

Nucleic acid target (also Nucleotide sequence that you want to detect.
called “target
template”)

Unknown sample The sample for which you want to determine the presence or
(also called sample of absence of a specific target.
interest)

How Plus/Minus Plus/minus assays begin by aliquoting the following to each well of a plate: PCR
Assays Work master mix, primers, and fluorogenic probes constructed for the target nucleic acid
sequence. Test samples are then added to the plate and loaded into a thermal cycler
for thermal cycling.
During the PCR, the fluorogenic probes anneal specifically to the complementary
target sequence between the forward and reverse primer sites on the template DNA.
Then during extension, AmpliTaq Gold® DNA polymerase cleaves the hybridized
probes in each sample containing the target. The cleavage of each matched probe
separates the reporter dye from the quencher dye, resulting in increased fluorescence
by the reporter.
After thermal cycling, the plate is run on a real-time PCR instrument, which reads
the fluorescence generated during the PCR amplification. The fluorescent signals,
measured by the Sequence Detection Systems (SDS) software, can determine the
presence or absence of the target nucleic acid in each sample on the plate.

Incorporating an IPC
An IPC is a second TaqMan probe and primer set added to the reaction plate to detect
a low-copy, constitutive nucleic acid. If a well does not exhibit amplification, the
SDS software uses the positive signal from the IPC to confirm that the well failed to
amplify because of a lack of template, rather than a pipetting error.

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Chapter 5 Plus/Minus Assays

The configuration of samples on a plus/minus assay plate is slightly different when

using an IPC. For plates containing an IPC, primers, probe, and template are pipetted
with the target assay to all unknown wells on the plate. In addition to the NTC and
unknown samples, a positive control for the IPC is also arrayed on the plate. This
group of wells (IPC+) contains IPC template, IPC primers and probe, target primers
and probe, but no target template.

Purchasing the Applied Biosystems TaqMan Exogenous

IPC Reagents Kit
The Applied Biosystems TaqMan Exogenous Internal Positive Control Reagents kit
contains a pre-optimized internal positive control (IPC) that can be spiked into
samples to distinguish true target negatives from PCR failure due to inhibition.

Kit Features The kit is designed to:

• Distinguish types of negative results:
– A negative call for the target sequence and positive call for the IPC indicates
that no target sequence is present.
– A negative call for the target sequence and negative call for the IPC suggests
PCR inhibition.
• Avoid amplification of endogenous genes
• Permit co-amplification of the IPC and the target sequence without
compromising amplification of the target sequence
• Perform optimally with the TaqMan® 2✕ Universal PCR Master Mix

Amplifying the By using the TaqMan Exogenous IPC Reagents, a low-copy target DNA can be
IPC and Target in amplified in the same tube with the IPC. Although the target and IPC DNAs may
the Same Tube differ in initial copy number, the amplification efficiency of the target reaction is not
compromised due to limiting concentrations of IPC primers in the PCR reaction.
In the PCR reaction, the IPC is detected using a VIC® dye-labeled probe, and the
target template is detected using a FAM™ dye labeled probe.

Endpoint The TaqMan Exogenous IPC Reagents are designed for endpoint (plate read) assays
Detection and only. Endpoint detection collects fluorescence data after PCR is complete.
Post-PCR Plate Note: To aid in troubleshooting plus/minus assays, you can use the 7500 Fast, 7500,
Read or 7300 Systems to perform Real-Time analysis of PCR. If using a Real-Time PCR
System for PCR amplification, perform the pre-read and post-read runs separately.

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 Purchasing the Applied Biosystems TaqMan Exogenous IPC Reagents Kit

Available Kits Note: The part numbers listed in Table 5-2 are for 200 reactions. See Appendix B for
a list of available kit sizes. The reagent configurations listed below are recommended
for plus/minus assays.

Table 5-2 Reagent configurations for plus/minus assays

Reagent Configuration Part Number

TaqMan Exogenous Internal Positive Control Reagents with TaqMan® 2✕ 4308320

Universal PCR Master Mix (with VIC dye)

TaqMan Exogenous Internal Positive Control Reagents 4308323

Note: If you are using this kit, you will need to purchase one of the core
reagents separately, as described below.
TaqMan® 2✕ Universal PCR Master Mix, 4304437
TaqMan PCR Core Reagents Kit, N808-0228

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Chapter 5 Plus/Minus Assays

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Troubleshooting 6 6
This chapter covers:
Troubleshooting Quantification Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2
Troubleshooting Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-7

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Chapter 6 Troubleshooting

Troubleshooting Quantification Assays

Using SDS When you have irregular data, you can use the Sequence Detection Systems (SDS)
Software to software to diagnose some chemistry- and instrument-related problems. Table 6-1
Diagnose describes how to verify the integrity of your run data and how to begin
Irregular Data troubleshooting problems.

Table 6-1 Troubleshooting analyzed run data: quantification assays

Data Display Analysis Criteria Recommended Action

Raw Data Plot • Signal tightness and uniformity: Do • Clean the contaminated block and/or
the raw spectra signals from generate new samples.
Displays the composite raw
replicate groups and controls • Repeat the experiment.
fluorescence signal
exhibit similar spectral profiles? If
(not normalized) for the
not, the plate or sample block could
selected wells during each
be contaminated.
cycle of PCR.
• Characteristic signal shape: Do the
samples peak at the expected
wavelengths? For example,
samples containing only FAM™ dye-
labeled TaqMan® probes should not
produce raw fluorescence in the
wavelength of a VIC® dye
component. A signal present from
wells that do not contain the dye
could indicate that the sample,
master mix, or wells contain
contaminants.

Characteristic signal growth: As you • Be sure to mix reaction components

drag the bar through the PCR cycles, thoroughly to produce a homogeneous
do you observe growth as expected? solution.
Absent growth curves may indicate a • Ensure pipettes are calibrated.
pipetting error (well lacks template) or
no amplification.

Signal plateaus: Do any of the signals • Repeat sample dilutions.

plateau? Signal plateaus or saturation • Ensure pipettes are calibrated.
can indicate that a well contains too
much template or fluorescence. The
7900HT instrument saturates at 66,000
fluorescent units.

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 Troubleshooting Quantification Assays

Table 6-1 Troubleshooting analyzed run data: quantification assays (continued)

Data Display Analysis Criteria Recommended Action

Multicomponent Plot Correct dyes displayed: Does the plot Ensure that the plate is set up and the
display all dyes as expected? The detectors are labeled correctly.
Displays a plot of normalized
presence of an unexpected dye may
multicomponent data from a
result from an error in detector setup,
single well of a real-time run.
such as assigning the wrong reporter
Displays the component dye
or quencher dye.
signals that contribute to the
composite signal for the well.
ROX™ dye fluorescence level: Is the • Mix reaction components thoroughly to
ROX dye fluorescence signal less produce a homogeneous solution.
intense than that of the reporter dyes? • Ensure pipettes are calibrated.
If not, the lack of reporter fluorescence
may be caused by an absence of • Check the seal of the optical adhesive
probe in the well (a pipetting error). cover for leaks.

A drop in the ROX dye fluorescence

level can be caused by a non-
homogenous sample mix, evaporation,
primer/ROX dye interaction, or
improperly seated cover.
Note: A slight drop in ROX dye may
occur after Cycle 35. This is normal.

Reporter dye fluorescence level: Is the • Mix reaction components thoroughly to

reporter dye fluorescence signal above produce a homogeneous solution.
background? The background signal is • Ensure pipettes are calibrated.
a measure of ambient fluorescence. If a
dye fails to fluoresce above the
background, the well is likely missing
probes labeled with the dye (well does
not contain probe, PCR master mix, or
both).

MSE Level: The MSE (mean squared Ensure that the plate is set up and the
error) is a mathematical representation detectors are labeled correctly.
of how accurately the
multicomponented data fit the raw
data. The higher the MSE value, the
greater the deviation of the
multicomponented data from the raw
data.

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Chapter 6 Troubleshooting

Table 6-1 Troubleshooting analyzed run data: quantification assays (continued)

Data Display Analysis Criteria Recommended Action

Amplification Plot Correct baseline and threshold • Identify the components of the
settings: Are the baseline and amplification curve. Determine the cycle
Displays data from real-time
threshold values set correctly? at which amplification first begins to be
runs after signal normalization
distinguishable from the noise, then set
and multicomponent analysis.
the baseline so that the endpoint is 1 to
Contains the tools for setting
2 cycles before this point. (Use the
the baseline and threshold
linear view of the amplification plot to
cycle (CT) values for the run.
determine baseline.)
• Identify the components of the
amplification curve and set the
threshold so that it is:
– Above the background
– Below the plateaued and linear
regions
– Within the geometric phase of the
amplification curve

Irregular amplification: Do all samples • Ensure the instrument lamp has not
appear to have amplified normally, with exceeded 2000 hours.
a smooth amplification plot free of • Mix reaction components thoroughly to
sharp spikes or dips? The three phases produce a homogeneous solution
of the amplification curve should be
clearly visible in from each well.

Outlying amplification: When the run Check the seal of the optical adhesive
data is viewed in the CT vs. Well cover for leaks.
Position plot, do replicate wells amplify
comparably? Wells producing CT
values that differ significantly from the
average for the associated replicate
wells may be considered outliers.
If a plate produces nonuniformity
between replicates, some samples on
the plate may have evaporated.

6-4
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 Troubleshooting Quantification Assays

Troubleshooting Chemistry problems are often the cause of abnormal results. For example, a curve
Chemistry shaped like an upside down U indicates that too much template is present. Table 6-2
Problems describes several chemistry problems that may be encountered when running
quantification assays.

Table 6-2 Troubleshooting chemistry problems: quantification assays

Observation Possible Cause Recommended Action

Poor amplification of target Poor quality template • Verify the purity of each template preparation
by agarose electrophoresis, ensuring that only
one product is formed.
• Ensure that the 260/280 ratio ≥ 1.8

Poor RT conversion to cDNA • Check the RNA sample for degradation.

• Input RNA could be too concentrated or too
dilute. Calculate serial dilutions of template
RNA from original stock, then repeat RT PCR.
• Ensure RT PCR setup has been performed
under the appropriate conditions to avoid
premature cDNA synthesis.

Poor amplification of target in Control is too abundant and is Optimize the primer and probe concentrations for
multiplex assay outcompeting the target the target reaction. Limit primer concentrations of
the more abundant control.
Different primer-limited TaqMan® endogenous
controls can be used for multiplex optimization
with TaqMan® Gene Expression Assays.
See “Primer Limiting in Multiplex Assays” on
page 3-28 of this document.

Probe degradation Mix and aliquot the probe and primers into single-
use aliquots to prevent degradation by freeze-
thaw cycles.

Poor amplification of target in Target is difficult to amplify • Increase the annealing/extension temperature
multiplex assay when using: to 62 °C.
• TaqMan® Fast Universal • Increase the annealing/extension time in the
PCR Master Mix (2✕), No thermal cycler protocol to 30 seconds.
AmpErase® UNG • If you do not obtain acceptable performance
• Default Fast thermal cycling by increasing both the annealing/extension
conditions temperature and time, assay reoptimization
may be required. Refer to “Primer Limiting in
Multiplex Assays” on page 3-28 of this
document for more information.

Amplification of the target in the Contamination of NTCs • Verify that this is true amplification by
NTCs inspecting the multicomponent view for
cleavage of the TaqMan probe.
• Repeat the assay using new components for
NTC reaction mix.
• Use AmpErase® UNG.
• Use no-RT controls to rule out genomic
contamination.

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Chapter 6 Troubleshooting

Table 6-2 Troubleshooting chemistry problems: quantification assays (continued)

Observation Possible Cause Recommended Action

Decrease in fluorescence of the Precipitation or degradation in When using the TaqMan PCR Core Reagent kit,
passive reference dye the TaqMan buffers be sure to mix the tubes well.

Use TaqMan® 2✕ Universal PCR Master Mix

(PN 4304437). Be sure to mix thoroughly to
produce a homogeneous solution.

Make sure the kits have been stored properly, per

instructions on the packaging, and have not
expired.

Interactions between primers Re-design the primers and/or probe.

and probe with the passive
reference dye that cannot be
compensated for by the
baseline subtraction

Standard curve: poor slope Assay Design Guidelines not Follow guidelines precisely. Optimize assay probe
followed concentrations.
Note: A slope value of –3.32 is
equal to approximately 100%
efficiency. Incorrect dilutions Repeat sample dilutions. Ensure pipettes are
calibrated.

Inhibitors present in the Verify extraction method and reprecipitate DNA or

reaction RNA.

Improper reaction conditions Follow Applied Biosystems recommended

(protocols must be followed thermal profile.
precisely)

Standard curve: bad correlation Incorrect baseline and Verify settings according to the user guide
coefficient threshold settings specific to your system.
Note: The best correlation
coefficient is 1.0. Improper pipetting Check calibration of the pipettes. Pipette more
than 5 µL of sample.

Incorrect dilutions Repeat sample dilutions.

6-6
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 Troubleshooting Allelic Discrimination Assays

Troubleshooting Allelic Discrimination Assays

Troubleshooting DNA Concentration Effects
Run Data Observation: Scattering of data points
Possible Causes: Very low DNA concentrations or variable concentrations.
Figure 6-1 shows the benefits of using relatively high DNA concentrations. Using
relatively high DNA concentrations yields large ∆Rn.
As can be seen in Figure 6-2 on page 6-8, at 1 ng, 5 ng, and 20 ng, within a DNA
amount the cluster is tight and easily scorable. The data presented in Figure 6-2 was
generated by running the samples at low to high concentrations. If all samples have
relatively high (at least 1 ng) DNA concentration, the clusters are scorable. When
samples have very low DNA concentrations, such as 0.01 and 0.1, clusters become
diffuse, making scoring difficult.

ng/RXN
ng/RXN

20
20
5
5
11

0.1
0.1
0.01
0.01

Figure 6-1 Effects of DNA concentration on ∆Rn

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Chapter 6 Troubleshooting

3.5
20ng
3 NTC
5ng
2.5

Normalized FAM
1ng
2 0.1ng
0.01ng
1.5

0.5

0
0 0.5 1 1.5 2 2.5 3
Normalized VIC

Figure 6-2 Effects of different DNA concentrations

Recommended action: Verify that all samples have relatively high concentrations.

Troubleshooting If you have irregular data, you can use the SDS software to diagnose some
Analyzed Run chemistry- and instrument-related problems. Table 6-3 is a summary of checks to
Data verify the integrity of your run data and to help you begin troubleshooting potential
problems.

Table 6-3 Troubleshooting analyzed run data: allelic discrimination assays

Data Display Analysis Criteria Recommended Action

Raw Data • Signal tightness and uniformity: Do Clean the contaminated block and generate
the raw spectra signals from new samples. Repeat the experiment.
Displays the composite replicate groups and controls
fluorescence signal (not exhibit similar spectral profiles? If
normalized) for the selected not, the plate or sample block
wells during each cycle of the could be contaminated.
PCR.
• Characteristic signal shape: Do the
samples peak at the expected
wavelengths?
• Signal plateaus: Do any of the
signals plateau? Signal plateaus or
saturation can indicate that a well
contains too much template or
fluorescence signal.

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Formulas A A
Comparative CT Method for Relative Quantification
Formula The amount of target, normalized to an endogenous control and relative to a
calibrator, is calculated by:
2 –∆∆CT

Derivation of the The equation that describes the exponential amplification of PCR is:
Formula
Xn = Xo × ( 1 + EX ) n

where:
• xn = number of target molecules at cycle n
• xo = initial number of target molecules
• Ex = efficiency of target amplification
• n = number of cycles
• Xo =initial number of target molecules
The threshold cycle (CT) indicates the fractional cycle number at which the amount
of amplified target reaches a specified threshold. Thus,

X T = X o × ( 1 + E X ) CT, X = K X

where:
• xT = threshold number of target molecules
• CT, X = threshold cycle for target amplification
• KX = constant
A similar equation for the endogenous control reaction is:

R T = R o × ( 1 + E R ) CT, R = K R

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Appendix A Formulas

where:
• RT = threshold number of reference molecules
• Ro = initial number of reference molecules
• ER = efficiency of reference amplification
• CT, R = threshold cycle for reference amplification
• KR = constant
Dividing XT by RT yields the following expression:

C
XT X o × ( 1 + E X ) T, X K X
------- = -------------------------------------------
- = ------- = K
RT Ro × ( 1 + ER )
C T, R KR

The exact values of XT and RT depend on a number of factors, including:

• Reporter dye used in the probe
• Sequence context effects on the fluorescence properties of the probe
• Efficiency of probe cleavage
• Purity of the probe
• Setting of the fluorescence threshold.
Therefore, the constant K does not have to be equal to 1.
Assuming efficiencies of the target and the reference are the same:
E X = ER = E

Xo C –C
------ × ( 1 + E ) T, X T, R= K
Ro

Or

∆C T
XN × ( 1 + E ) =K

where:
• XN = XO/RO, the normalized amount of target
• ∆CT = CT, X – CT, R, the difference in threshold cycles for target and reference

A-2
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 Comparative CT Method for Relative Quantification

Rearranging gives the following expression:

– ∆C T
XN = K × ( 1 + E )

The final step is to divide the XN for any sample (q) by the XN for the calibrator (cb):

X – ∆C T, q
N, q K × ( 1 + E ) – ∆∆C T
-------------- = -------------------------------------------- == ( 1 + E )
X N, cb – ∆ C
K × ( 1 + E ) T, cb

where:
• ∆∆CT = ∆CT, q – ∆CT, cb
For amplicons designed and optimized according to Applied Biosystems assay
design guidelines (amplicon size < 150 bp), the efficiency is close to 1. Therefore,
the amount of target, normalized to an endogenous control and relative to a
calibrator, is given by:
2 –∆∆CT

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Appendix A Formulas

A-4
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Part Numbers B B
This chapter covers:
Real-Time PCR Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-2
Sequence Detection System Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-3
Real-Time PCR Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-4
Real-Time PCR Reagent Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-5
Real-Time PCR RT-PCR Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-7
Real-Time PCR Reaction Kits (with Controls) . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-8
Real-Time PCR Control Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-9
Real-Time PCR Reagent Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-10
Real-Time PCR Calibration Kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-11
Real-Time PCR Disposables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-12
TaqMan Pre-Developed Assay Reagents for Allelic Discrimination . . . . . . . . . .B-13
Custom Oligonucleotide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-14

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Appendix B Part Numbers

Real-Time PCR Instruments

Part Number Instrument

4329002 Applied Biosystems 7900HT Fast Real-Time PCR System

with Standard 384-Well Block Module and Automation
Accessory

4329004 Applied Biosystems 7900HT Fast Real-Time PCR System

with Standard 96-Well Block Module and Automation
Accessory

4329001 Applied Biosystems 7900HT Fast Real-Time PCR System

with Standard 384-Well Block Module

4329003 Applied Biosystems 7900HT Fast Real-Time PCR System

with Standard 96-Well Block Module

4351405 Applied Biosystems 7900HT Fast Real-Time PCR System

with Fast 96-Well Block Module

4351404 Applied Biosystems 7900HT Fast Real-Time PCR System

with Fast 96-Well Block Module and Automation Accessory

4351412 7900HT System Fast Service Upgrade

4329007 7900HT System Automation Accessory Upgrade

4329012 7900HT TaqMan® Low Density Array Upgrade

4331406 7900HT System Standard 384-Well Block Upgrade Kit

4331405 7900HT System Standard 96-Well Block Upgrade Kit

4351101 Applied Biosystems 7300 Real-Time PCR System (with

laptop computer)

4351103 Applied Biosystems 7300 Real-Time PCR System (with tower

computer)

4351104 Applied Biosystems 7500 Real-Time PCR System (with

laptop computer)

4351105 Applied Biosystems 7500 Real-Time PCR System (with tower

computer)

4351106 Applied Biosystems 7500 Fast Real-Time PCR System (with

laptop computer)

4351102 Applied Biosystems 7500 Fast Real-Time PCR System (with

tower computer)

4362143 7500 Fast Real-Time PCR Upgrade Kit

4349132 ABI PRISM ® HID 7000 SDS For Human Identification (with
laptop computer)

4349117 ABI PRISM ® HID 7000 SDS For Human Identification (with
tower computer)

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 Sequence Detection System Software

Sequence Detection System Software

Part Number Software Instrument

Sequence Detection System Software

Contact your 7900HT System Applied Biosystems 7900HT Fast

Applied Biosystems Software v.2.2.1 or Real-Time PCR System
sales representative. higher
ABI PRISM® 7900HT Sequence
Detection System

7300/7500/7500 Fast Applied Biosystems 7300 Real-Time

System Software v1.3 PCR System
or higher
Applied Biosystems 7500 Real-Time
PCR System

Applied Biosystems 7500 Fast Real-

Time PCR System

Associated Applied Biosystems Software

Part Number Software

Contact your Applied Primer Express® Software v3.0 or higher

Biosystems sales
representative.

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May 11, 2005 12:44 pm, AppB Part Numbers.fm

Appendix B Part Numbers

Real-Time PCR Kits

Part Number Kit Description Instrument

4307266 TaqMan® Cytokine Gene Expression Plate 1 with TaqMan® 2✕ Applied Biosystems
Universal PCR Master Mix and Control Total RNA 7900HT Fast Real-Time
PCR System,
Two MicroAmp® Optical 96-Well Reaction Plates pre-loaded with
Applied Biosystems
TaqMan primers and probes for 12 human cytokine targets (replicates
7300/7500 Real-Time
of eight) and the 18S Ribosomal RNA endogenous control (in all
PCR System
96 wells). TaqMan primer and probe concentrations are optimized for
multiplex PCR utilizing FAM™ and VIC® dyes. Configuration includes
TaqMan® 2✕ Universal PCR Master Mix, MicroAmp Optical Caps,
Control Total RNA (Human), and Protocol.

4307265 TaqMan® Cytokine Gene Expression Plate 1 with TaqMan® 2✕ Applied Biosystems
Universal PCR Master Mix 7900HT Fast Real-Time
PCR System,
Two MicroAmp Optical 96-Well Reaction Plates pre-loaded with
Applied Biosystems
TaqMan primers and probes for 12 human cytokine targets (replicates
7300/7500 Real-Time
of eight) and the 18S Ribosomal RNA endogenous control (in all
PCR System
96 wells). TaqMan primer and probe concentrations are optimized for
multiplex PCR utilizing FAM and VIC dyes. Configuration includes
TaqMan® 2✕ Universal PCR Master Mix and MicroAmp Optical Caps.

4306744 TaqMan® Cytokine Gene Expression Plate 1 Protocol —

4309920 TaqMan® Human Endogenous Control Plate with TaqMan® 2✕ Applied Biosystems
Universal PCR Master Mix and Control Total RNA 7900HT Fast Real-Time
PCR System,
Two MicroAmp Optical 96-Well Reaction Plates pre-loaded with
Applied Biosystems
TaqMan primers and probes for 11 human endogenous control targets
7300/7500 Real-Time
and an internal positive control (IPC) in replicates of eight. TaqMan
PCR System
primer and probe concentrations are optimized for 50µl reactions and
utilize VIC dye. Configuration includes TaqMan® 2✕ Universal PCR
Master Mix, MicroAmp Optical Caps, Control Total RNA (Human), and
Protocol.

4309921 TaqMan® Human Endogenous Control Plate with TaqMan® 2✕ Applied Biosystems
Universal PCR Master Mix 7900HT Fast Real-Time
PCR System,
Two MicroAmp Optical 96-Well Reaction Plates pre-loaded with
Applied Biosystems
TaqMan primers and probes for 11 human endogenous control targets
7300/7500 Real-Time
and an internal positive control (IPC) in replicates of eight. TaqMan
PCR System
primer and probe concentrations are optimized for 50µl reactions and
utilize VIC dye. Configuration includes TaqMan® 2✕ Universal PCR
Master Mix and MicroAmp Optical Caps.

4308134 TaqMan® Human Endogenous Control Plate Protocol —

4352407 Fast Reagents Starter Kit Applied Biosystems

7500 Fast Real-Time
Three tubs containing TaqMan® 2X Universal PCR Master Mix, Control
PCR System
Total RNA (Human), and 18S TaqMan® Gene Expression Assay.

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 Real-Time PCR Reagent Kits

Real-Time PCR Reagent Kits

Part Number Kit Description Number of Reactions

4352042 TaqMan® Fast Universal PCR Master Mix (2✕), No AmpErase® UNG 250

4304437 TaqMan® 2✕ Universal PCR Master Mix 200

Supplied at a 2✕ concentration. The mix is optimized for
TaqMan® reactions and contains AmpliTaq Gold® DNA Polymerase,
AmpErase® UNG, dNTPs with dUTP, Passive Reference 1, and
optimized buffer components. One 5-mL vial in each box.

4318157 TaqMan® 2✕ Universal PCR Master Mix 2000

Ten 5-mL vials in each box.

4305719 TaqMan® 2✕ Universal PCR Master Mix 10-Pack 2000

Ten boxes of PN 4304437.

4326708 50 mL, TaqMan® 2✕ Universal PCR Master Mix 2000

4324018 TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG 200

Supplied at a 2✕ concentration. The mix is optimized for
TaqMan® reactions and contains AmpliTaq® Gold DNA Polymerase,
dNTPs with dUTP, Passive Reference 1, and optimized buffer
components.
One 5-mL vial in each box.

4324020 TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG 10- 2000
Pack
Ten 5-mL vials in each box.

4326614 TaqMan® 2✕ Universal PCR Master Mix, No AmpErase® UNG 50 ml 2000

4304449 TaqMan® 2✕ Universal PCR Master Mix Protocol —

N808-0228 TaqMan® PCR Core Reagents Kit 200

250 Units AmpliTaq® Gold DNA Polymerase, 100 Units
AmpErase® UNG, dUTP, dATP, dCTP, dGTP, 10✕ TaqMan Buffer A, 25
mM MgCl2 Solution.

4304439 TaqMan® 1000 RXN PCR Core Reagents 1000

1250 Units AmpliTaq® Gold DNA Polymerase, 500 Units
AmpErase® UNG, dUTP, dATP, dCTP, dGTP, 10✕ TaqMan Buffer A, 25
mM MgCl2 Solution.

402930 TaqMan® PCR Core Reagent Kit 10-Pack 2000

Ten of PN N808-0228.

402823 TaqMan® PCR Reagent Kit Protocol —

4304886 SYBR® Green PCR Core Reagents 200

250 Units AmpliTaq®Gold DNA Polymerase, 100 Units
AmpErase® UNG, dNTP Mix with dUTP, 10✕ SYBR Green PCR Buffer,
25 mM MgCl2 Solution.

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Appendix B Part Numbers

4306736 10-Pack, SYBR® Green PCR Core Reagents 2000

Ten of PN 4304886.

4304965 SYBR® Green PCR Core Reagents Protocol —

4309155 SYBR® Green PCR Master Mix 200

Supplied at a 2✕ concentration. The mix is optimized for
SYBR® Green reagent reactions and contains SYBR® Green 1 dye,
AmpliTaq® Gold DNA Polymerase, dNTPs with dUTP, Passive
Reference 1, and optimized buffer components. One 5-mL vial in each
box.

4312704 SYBR® Green PCR Master Mix 10-Pack 2000

Ten of PN 4309155.

4334973 SYBR® Green PCR Master Mix 2000

4334973 SYBR® Green PCR Master Mix, 50-mL 2000

4344463 SYBR® Green PCR Master Mix, 1-mL 40

4310251 SYBR® Green PCR Master Mix Protocol —

Combined protocol for SYBR® Green PCR Master Mix and
SYBR® Green RT-PCR Reagents.

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 Real-Time PCR RT-PCR Kits

Real-Time PCR RT-PCR Kits

Number of 50-µL
Part Number Kit Description
Reactions

4309169 TaqMan® One-Step RT-PCR Master Mix Reagents Kit 200

®
Vial1: AmpliTaq Gold DNA Polymerase mix (2✕) is optimized for
TaqMan® reactions and contains AmpliTaq® Gold DNA Polymerase,
dNTPs with dUTP, Passive Reference 1, and optimized buffer
components. Vial 2: RT enzyme mix (40✕) contains
MultiScribe™ Reverse Transcriptase and RNase Inhibitor.
4313803 TaqMan® One-Step RT-PCR Master Mix Reagents Kit 10-Pack 2000
Ten of PN 4309169

4310299 TaqMan® One-Step RT-PCR Master Mix Reagents Kit Protocol —

N808-0232 TaqMan® Gold RT-PCR Reagents without controls 200
TaqMan® PCR Core Reagents Kit (N808-0228), TaqMan® Reverse
Transcriptase Reagents (N808-0234).
4304133 TaqMan® Gold RT-PCR Reagents without controls 10-Pack 2000
Ten of PN N808-0232
402876 TaqMan® Gold RT-PCR Protocol —
®
N808-0234 TaqMan Reverse Transcriptase Reagents 200
™
MultiScribe Reverse Transcriptase, RNase Inhibitor, dNTP Mixture,
Oligo d(T)16, Random Hexamers, 10✕ RT Buffer, MgCl2 Solution.
4304134 TaqMan® Reverse Transcriptase Reagents 10-Pack 2000
Ten of PN N808-0234
N808-0236 TaqMan® EZ RT-PCR Core Reagents 200
1000 Units rTth DNA Polymerase, 100 Units AmpErase® UNG, dUTP,
dATP, dCTP, dGTP, 5✕ TaqMan® EZ Buffer, 25 mM Mn(OAc)2.
403028 TaqMan® EZ RT-PCR Core Reagents 10-Pack 2000
Ten of PN N808-0236
402877 TaqMan® EZ RT-PCR Kit Protocol —
4310179 SYBR® Green RT-PCR Reagents 200
SYBR® Green PCR Master Mix (4309155), TaqMan® Reverse
Transcriptase Reagents (N808-0234).
4310251 SYBR® Green PCR Master Mix Protocol —
Combined protocol for SYBR®
Green PCR Master Mix and
SYBR® Green RT-PCR Reagents.
4322171 High-Capacity cDNA Archive Kit 200
Random primers, Optimized RT Buffer, dNTPs and MultiScribe™
MULV/RNase Inhibitor mix for the conversion of up to 10 µg of total
RNA in a single 100-µL reaction to single-stranded cDNA.
4322169 High-Capacity cDNA Archive Kit Protocol —

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Appendix B Part Numbers

Real-Time PCR Reaction Kits (with Controls)

Part Number Kit Description Number of Reactions

N808-0230 TaqMan® PCR Reagent Kit 200

TaqMan PCR Core Reagents Kit, β-actin control reagents, and
®

Protocol.

N808-0233 TaqMan® Gold RT-PCR Reagents with controls 200

® ®
TaqMan PCR Core Reagent Kit, TaqMan Reverse Transcriptase
Reagents, TaqMan® GAPDH Control Reagents, and Protocol.

N808-0235 TaqMan® EZ RT-PCR Kit 200

1000 Units rTth DNA Polymerase, 100 Units AmpErase® UNG, dUTP,
dATP, dCTP, dGTP, TaqMan® EZ Buffer, Mn(OAc)2, 100 GAPDH control
reactions, and Protocol.

N808-0236 TaqMan® EZ RT-PCR Core Reagents Without Controls 200

403028 TaqMan® EZ-RT-PCR Core Reagents, 10-pack 2000

4352407 TaqMan® Fast Reagents Starter Kit 500

®
One tube TaqMan Fast Universal PCR Master Mix (2✕), No
AmpErase® UNG, one tube Human Control cDNA, one tube 20✕ 18s
rRNA.

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 Real-Time PCR Control Reagents

Real-Time PCR Control Reagents

Part Number Kit Description Number of Reactions

4308329 TaqMan® Ribosomal RNA Control Reagents (VIC® Dye) 1000

®
Human Control RNA, rRNA Probe (VIC dye), rRNA Forward Primer, and rRNA
Reverse Primer.
4308310 TaqMan® Ribosomal RNA Control Reagents Protocol (VIC® Dye) —

4316844 TaqMan® RNase P Control Reagents (VIC® Dye) 1000

20✕ primer and probe (VIC® dye) mix and Human Genomic Control DNA.
4308313 TaqMan® Rodent GAPDH Control Reagents (VIC® Dye) 1000
Rodent Control RNA, rodent GAPDH Probe (VIC® dye), rodent GAPDH
Forward Primer, and rodent GAPDH Reverse Primer.
4308318 TaqMan® Rodent GAPDH Control Reagents Protocol (VIC® Dye) —
402869 TaqMan® GAPDH Control Reagents (Human) 100
Human Control RNA, GAPDH Probe (JOE™ dye), GAPDH Forward Primer, and
GAPDH Reverse Primer.
401846 TaqMan® β-actin Detection Reagents 100
401970 TaqMan® DNA Template Reagents —
4308323 TaqMan® Exogenous Internal Positive Control Reagents (VIC® Dye) 200
®
10✕ exogenous IPC primer and probe (VIC dye) mix, 10✕ exogenous IPC
blocking reagent, and 50✕ exogenous IPC target. Kit represents an IPC
assay (optimized with TaqMan® 2✕ Universal PCR Master Mix) to be used with
custom + / - assays.
4308321 5-Pack, TaqMan® Exogenous IPC Reagents (VIC® Dye) 1000
Five of PN 4308323
4308320 TaqMan® Exogenous Internal Positive Control Reagents (VIC® Dye) with 200
TaqMan® 2✕ Universal PCR Master Mix
(PN 4308323 and 4304437)
4308335 TaqMan® Exogenous Internal Positive Control Reagents Protocol (VIC® Dye) —
4316831 TaqMan® RNase P Detection Reagents Kit 100
20✕ primer and probe (FAM™ dye) mix and Human Genomic Control DNA.

4310982 TaqMan® RNase P Instrument Verification Plate One 96-Well Plate

®
One ABI PRISM 96-Well Optical Reaction Plate pre-loaded and sealed with
TaqMan® primers and probe to detect and quantitate genomic copies of the
human RNase P gene.
4351979 TaqMan® RNase P Fast 96-Well Instrument Verification Plate One Optical 96-Well
Fast Plate
One Optical 96-Well Fast Thermal Cycling Plate with Barcode (code 128) pre-
loaded and sealed with TaqMan® primers and probe to detect and quantitate
genomic copies of the human RNase P gene.

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Appendix B Part Numbers

4323306 TaqMan® RNase P 384-Well Instrument Verification Plate One 384-Well Plate
One ABI PRISM ® 384-Well Optical Reaction Plate preloaded and sealed with
complete TaqMan® primers and probe to detect and quantitate genomic
copies of the human RNase P gene.

Real-Time PCR Reagent Components

Part Number Description Quantity

4304441 TaqMan® 1000 RXN Gold with Buffer A Pack 1000 Reactions
® ®
1250 Units AmpliTaq Gold DNA Polymerase, 10✕ TaqMan Buffer A,
25 mM MgCl2 Solution.

4305822 Sequence Detection Systems Spectral Calibration Kit —

4311235 MultiScribe™ Reverse Transcriptase, 100-µL 5000 Units

(400 Rxn @ 10 µL each, 40 Rxn @ 100 µL)

N808-0119 RNase Inhibitor 2000 Units

N808-0260 dNTP Mixture (10 mM) 1 mL

N808-0128 Oligo d(T)16 (50 µM) 0.1 mL

N808-0127 Random Hexamers (50 µM) 0.1 mL

4307281 Control Total RNA (Human) 100 µL

(50 ng/µL)

N808-0096 AmpErase® Uracil N-glycosylase (UNG) 100 µL

(1 unit/µL)

4312660 Control Total DNA (Human) 2 tubes, each 100 µL

(10 ng/µL)

402929 20% Glycerol Solution, Molecular Biology Grade 100 mL

B-10
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 Real-Time PCR Calibration Kits

Real-Time PCR Calibration Kits

Part Number Kit Description Quantity

4305822 Sequence Detection Systems Spectral Calibration Kit —

4323977 Sequence Detection Systems 384-Well Spectral Calibration Kit Two 384-Well
Plates
Two ABI PRISM ® 384-Well Optical Reaction Plates. One preloaded and
sealed Background plate and one preloaded and sealed Spectral
Calibration plate with eight separate dye standards (FAM™, JOE™, NED™,
ROX™, SYBR® Green, TAMRA™, TET™, and VIC® dyes).

4351653 7900HT System Fast 96-Well Spectral Calibration Kit Three Optical
96-Well Fast Plates
Three Optical 96-Well Fast Thermal Cycling Plates with Barcode (code 128):
one preloaded and sealed Background plate and two preloaded and sealed
Spectral Calibration plates containing eight separate dye standards (FAM™,
JOE™, NED™, ROX™, SYBR® Green, TAMRA™, TET™, and VIC® dyes).

4328895 ABI PRISM ® 7000 Sequence Detection Systems Spectral Calibration Kit Eight Optical
96-Well Reaction
Eight ABIPRISM ® Optical 96-Well Reaction Plates. One preloaded and
Plates
sealed Background plate and seven sealed and preloaded Spectral
Calibration plates containing seven separate dye standards (FAM™, JOE™,
NED™, ROX™, SYBR® Green, TAMRA™, and VIC® dyes).

4350584 7300/7500 Real-Time PCR Systems TaqMan® RNase P 96-Well Instrument 1 kit
Verification Plate

4348187 Applied Biosystems 7300 Real-Time PCR System Spectral Calibration Kit Nine Optical
96-Well Reaction
Nine Optical 96-Well Reaction Plates. One preloaded and sealed
Plate
Background plate, seven preloaded and sealed Pure Dye plates containing
seven different dye standards (FAM™, JOE™, NED™, ROX™, SYBR® Green,
TAMRA™, and VIC® dyes), and one preloaded and sealed ROI Calibration
plate.

4349180 Applied Biosystems 7500 Real-Time PCR System Spectral Calibration Kit I Nine Optical
96-Well Reaction
Nine Optical 96-Well Reaction Plates. One preloaded and sealed
Plates
Background plate, seven preloaded and sealed Pure Dye plates containing
ten different dye standards (FAM™, JOE™, NED™, ROX™, SYBR® Green,
TAMRA™, and VIC® dyes), and one preloaded and sealed ROI Calibration
plate.

4351151 Applied Biosystems 7500 Real-Time PCR System Spectral Calibration Kit II Five Optical
96-Well Reaction
Five Optical 96-Well Reaction Plates. One preloaded and sealed
Plates
Background plate, three preloaded and sealed Pure Dye plates containing
ten different dye standards (CY3™, CY5™, and TEXAS RED® dyes), and one
preloaded and sealed ROI Calibration plate.

4360788 Applied Biosystems 7500 Fast Real-Time PCR System Spectral Calibration Twelve Optical
Kit I 96-Well Fast Plates
Twelve Optical 96-Well Fast Thermal Cycling Plates. One preloaded and
sealed Background plate, ten preloaded and sealed Pure Dye plates
containing ten different dye standards (CY3™, CY5™, FAM™, JOE™, NED™,
ROX™, SYBR® Green, TAMRA™, TEXAS RED®, and VIC® dyes), and one
preloaded and sealed ROI Calibration plate.

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May 11, 2005 12:44 pm, AppB Part Numbers.fm

Appendix B Part Numbers

4362201 Applied Biosystems 7500 Fast Real-Time PCR System Spectral Calibration
Kit II

4351979 TaqMan® RNase P Chemistry Installation Kit (7500 Fast System)

Real-Time PCR Disposables

Part Number Item Quantity

4313663 ABI PRISM ® Optical Adhesive Cover Starter Kit 20 Covers/Pkg

ABI PRISM ® Optical Adhesive Covers (quantity 20), Applicator
(quantity 1), ABI PRISM ® Optical Cover Compression Pad (quantity 1)

4311971 ABI PRISM ® Optical Adhesive Covers 100 Covers/Pkg

4314320 ABI PRISM ® Optical Adhesive Covers and ABI PRISM ® 96-Well Optical 100 Covers
Reaction Plate with Barcode (code 128)
100 Plates/Pkg
PN 4311971 and 5✕ ABI PRISM ® 96-Well Optical Reaction Plates
(PN 4306737)

4360954 Optical Adhesive Covers 25 Covers/Pkg

4312639 ABI PRISM ® Optical Cover Compression Pads 5 Pads/Pkg

4309849 ABI PRISM ® 384-Well Clear Optical Reaction Plate with Barcode 50 Plates/Pkg
(code 128)

4326270 ABI PRISM ® 384-Well Clear Optical Reaction Plate with Barcode (code 500 Plates/Pkg
128),10-Pack
10✕ (PN 4309849) ABI PRISM ® 384-Well Clear Optical Reaction Plate
with Barcode (code 128).

4306737 ABI PRISM ® 96-Well Optical Reaction Plate with Barcode (code 128) 20 Plates/Pkg

4326659 ABI PRISM ® 96-Well Optical Reaction Plate with Barcode (code 128), 500 Plates/Pkg
25-Pack
25✕ (PN 4306737) ABI PRISM ® 96-Well Optical Reaction Plate with
Barcode (code 128).

4323032 ABI PRISM ® Optical Caps, 8 Caps/Strip 300 Strips/Pkg

Flat Caps. 2400 Caps/Pkg

4330015 ABI PRISM ® Cap Installing Tool One/Pkg

® ®
Used for removing MicroAmp Caps or ABI PRISM Optical Caps from
the MicroAmp® plate or ABI PRISM ® 96-Well Optical Reaction Plate
and Tray/Retainer Assemblies.

B-12
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 TaqMan Pre-Developed Assay Reagents for Allelic Discrimination

4333292 ABI PRISM ® Snap-On Compression Pads 9/Pkg

Snap-On Compression Pad is a compliant cover bonded to a metal
frame, and is designed for use on the Applied Biosystems 7900HT
Fast Real-Time PCR System when using the available automation
accessory with 96-well plates.

4333183 Adhesive Seal Applicators 5/Pkg

N801-0560 MicroAmp® Optical 96-Well Reaction Plate 10 Plates/Pkg

Not Barcoded.

403012 MicroAmp® Optical 96-Well Reaction Plates and ABI PRISM ® Optical 20 Plates
Caps
PN 4306737 and 4323032. 2400 Caps/Pkg

4312063 MicroAmp® Splash Free Support Base for 96-Well Reaction Plates 10 Bases/Pkg

4346906 Optical 96-Well Fast Thermal Cycling Plate with Barcode (code 128) 20 plates

4346907 Optical 96-Well Fast Thermal Cycling Plate without Barcode 20 plates

TaqMan Pre-Developed Assay Reagents for Allelic

Discrimination
Quantity
Part Number TaqMan® PDAR Description
(Number of Reactions)

4312561 CYP2C19*2 400 reactions

4312562 CYP2C19*3 400 reactions

4312559 CYP2C9*2 400 reactions

4312560 CYP2C9*3 400 reactions

4312554 CYP2D6*3 400 reactions

4312555 CYP2D6*4 400 reactions

4312556 CYP2D6*6 400 reactions

4312557 CYP2D6*7 400 reactions

4312558 CYP2D6*8 400 reactions

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May 11, 2005 12:44 pm, AppB Part Numbers.fm

Appendix B Part Numbers

Custom Oligonucleotide Synthesis

Part Number Item Delivery Time a

4316034 TaqMan® MGB Probe 6,000 pmoles 6 to 7 days

™ ® ™ ™
5′-Fluorescent dye label: 6-FAM , VIC , NED , or TET dyes.

4316033 TaqMan® MGB Probe 20,000 pmoles 6 to 7 days

™ ® ™ ™
5′-Fluorescent dye label: 6-FAM , VIC , NED , or TET dyes.

4316032 TaqMan® MGB Probe 50,000 pmoles 6 to 7 days

™ ® ™ ™
5′-Fluorescent dye label: 6-FAM , VIC , NED , or TET dyes.

450025 TaqMan® TAMRA™ Probe 6,000 pmoles 4 to 5 days

™ ® ™
5′-Fluorescent dye label: 6-FAM , VIC , or TET dyes.

450024 TaqMan® TAMRA™ Probe 20,000 pmoles 4 to 5 days

™ ® ™
5′-Fluorescent dye label: 6-FAM , VIC , or TET dyes.

450003 TaqMan® TAMRA™ Probe 50,000 pmoles 4 to 5 days

™ ® ™
5′-Fluorescent dye label: 6-FAM , VIC , or TET dyes.

4304970 Sequence Detection Primers: 10,000 pmoles 3 days

Minimum 4,000 pmoles purified for sequence detection.

4304971 Sequence Detection Primers: 80,000 pmoles 4 days

Minimum 40,000 pmoles purified for sequence detection.

4304972 Sequence Detection Primers: 130,000 pmoles 4 days

Minimum 130,000 pmoles purified for sequence detection.

a. For custom oligonucleotide synthesis, consider the following:

– Delivery includes only business week days; orders to be received by email or online web ordering before 10:00 a.m.
PST/PDT.
– Orders received by fax are entered into the order system on the next business day.
– Quantities are based on an average 23-mer oligo length.
Contact information:
– To order online: http://store.appliedbiosystems.com
– To order by email: OligosUS@appliedbiosystems.com

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References C C
Afonina, I., Zivarts, M., Kutyavin, I., et al. 1997. Efficient priming of PCR with
short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res.
25:2657–2660.
Collins, M. L., Zayati, C., Detmar, J. J., Daly, B., Kolberg, J. A. Cha, T. A., Irvine, B.
D. Tucker, J., and Urdea, M. S. 1995. Preparation and characterization of RNA
standards for use in quantitative branched DNA hybridization assays. Anal. Biochem.
Mar 20 226:120–129.
Förster, V. T. 1948. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann.
Physics (Leipzig) 2:55–75.
Higuchi, R., Dollinger, G., Walsh, P.S., and Griffith, R. 1992. Simultaneous
amplification and detection of specific DNA sequences. Biotechnology 10:413–417.
Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR:Real time
monitoring of DNA amplification reactions. Biotechnology 11:1026–1030.
Kutyavin, I.V., Lukhtanov, E.A., Gamper, H.B., and Meyer, R.B. 1997.
Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base
composition and backbone effects on hybridization. Nucleic Acids Res.
25:3718–3723.
Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR. Nature
339:237–238.
Lee, L. G., Connell, C. R., and Block, W. 1993. Allelic discrimination by nick-
translation PCR with fluorogenic probes. Nucleic Acids Res. 21:3761–3766.
Livak, K.J., and Schmittgen, T.D. 2001. Analysis of Relative Gene Expression Data
Using Real-Time Quantitative PCR and the 2–∆∆CT Method. Methods 25:402–408.
Livak, K.J., Flood, S.J.A., Marmaro, J.,Guisti W., Deetz, K. 1995. Oligonucleotides
with fluorescent dyes at opposite ends provide a quenched probe system useful for
detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4:357–62.
Livak, K.J., Flood, S.J.A., Marmaro, J., and Mullah, K.B., inventors; Perkin-Elmer
Corporation (Foster City, CA), assignee. 2 Mar. 1999. Hybridization assay using self-
quenching fluorescence probe. United States patent 5,876,930.
Livak, K.J., Marmaro, J., and Todd, J.A. 1995. Towards fully automated genome-
wide polymorphism screening [letter]. Nat. Genet. 9:341–342.
Longo, M.C., Berninger, M.S., and Hartley, J.L. 1990. Use of uracil DNA
glycosylase to control carry-over contamination in polymerase chain reactions. Gene
93:125–128.

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Appendix C References

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Index

Symbols assay design

considerations 3-10
guidelines 1-8
Primer Express software 3-10
Numerics assay source, selecting 1-7
3’ primer end, and amplicon sites 3-12 assay type
5’ nuclease quantification assays 3-22 supported types 1-2
using SYBR Green I chemistry 1-6
5’ probe end, and amplicon sites 3-12 using TaqMan probe-based chemistry 1-6

A B
absolute quantification
biohazardous waste, handling xv
standard curve 3-53
troubleshooting 6-7 biological hazard guidelines xvi
absolute standard curve, performing 3-53 bold text, when to use vii
allelic discrimination assay
category 1-2 C
conclusions 4-7 calculation methods, absolute quantification 3-34
described 4-2
designing your own 4-6 calibrator, for sample comparisons 3-40
guidelines for probes 4-7 carryover, UNG to minimize 2-6
how it works 4-3 CAUTION, description xii
instruments 4-2 chemical safety xiii, xiv
mismatches 4-3 chemical waste safety xiv, xv
optimizing 4-6
chemistries, selecting 1-6
Primer Express software 4-7
reagent configurations 4-9 chemistry choice
TaqMan MGB probes 4-7 assay type 2-5
TaqMan probe-based chemistry, and 4-2 quantification assays 2-5
terms defined 4-2 selection criteria 2-5
thermal cycling parameters 4-10 coefficient of variation, calculating 3-39
troubleshooting 6-7 comparative CT
amplicon sites example 3-44
3’ primer end, and 3-12 formula 3-42
5’ probe end, and 3-12 how used 3-45
and melting temperature 3-12 multiplex PCR 3-50
G/C content 3-12 performing 3-44
screening 3-11 relative quantification 3-42
selection 3-11 components, Real-Time PCR reagents B-10
amplicons, selecting small 3-11 contamination, minimizing DNA 2-6
analyzed run data, troubleshooting allelic conventions
discrimination 6-8 bold text vii
Applied Biosystems IMPORTANTS! vii
contacting ix in this guide vii
customer feedback on documentation viii italic text vii
Services and Support ix menu commands vii
Technical Communications viii Notes vii
Technical Support ix safety xii

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May 11, 2005 12:43 pm, ChemGuideIX.fm

 user attention words vii I
CT calculation, relative efficiency 3-44
IMPORTANT, description xii
custom oligonucleotide synthesis B-14
IMPORTANTS!, description vii
Custom TaqMan Assays
instruments, Real-Time PCR B-2
about 1-7
Genotyping and Gene Expression 1-7 internal positive control, plus/minus assay 5-2
Custom TaqMan Gene Expression Assays 3-9 IPC
incorporating 5-3
Custom TaqMan SNP Genotyping Assays 4-6
plus/minus assay 5-2
customer feedback, on Applied Biosystems
italic text, when to use vii
documents viii
cv, calculating 3-39
K
D kits
Real-Time PCR B-4
DANGER, description xii Real-Time PCR calibration B-11
data analysis, gene expression 3-32 Real-Time PCR reaction, controls B-8
design guidelines Real-Time PCR reagent B-5
assay 1-8 RT-PCR B-7
primer and probe 3-13
quantification assays 3-10 M
disposables, Real-Time PCR B-12
melting temperature, and amplicon sites 3-12
DNA/cDNA quantification, thermal-cycling
parameters 3-17 menu commands, conventions for describing vii
documentation mismatch, in allelic discrimination assay 4-3
feedback viii MSDSs
related to this guide viii description xiii
dye-binding obtaining xiii
methods of 2-2 MSDSs, obtaining ix
requirements for Real-Time PCR 2-2 multicomponenting 3-47
SYBR Green I dye 2-2 multiple reporter dyes 3-47
multiplex accuracy, reporter dyes 3-47
E multiplex assays, rRNA primers 3-28
efficiency, target and reference 3-44 multiplex PCR
endogenous control, and standardizing 3-36 and relative quantification 3-45
comparative CT example 3-50
endpoint assay
comparative CT results 3-51
allelic discrimination 4-2 described 1-5
category 1-2
multiple reporter dyes 3-47
described 1-5 primer limiting 3-28
relative standard curve 3-48
G relative standard curve example 3-48
singleplex comparison 3-27
G/C content, and amplicon sites 3-12
using 3-27
gene expression, data analysis process 3-32
MultiScribe reverse transcriptase, defined 3-16
guidelines
chemical safety xiv
chemical waste disposal xiv N
chemical waste safety xv no amplification control
guidelines, designing an assay 1-8 See also NAC 5-3
nonspecific product, contamination with SYBR
H dye 2-6
Notes, description vii
hairpin loops, and primer choice 3-7
hazard symbols. See safety symbols, on instruments
hazards. See safety

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O 5’ nuclease 3-22
category 1-2
one-step RT-PCR, and RNA quantification 3-15 design guidelines 3-10
one-step RT-PCR, primers used 3-6 explained 3-4
optimization instruments used 3-4
allelic discrimination assays 4-6 optimizing SYBR Green I Dye 3-23
probe concentration 3-24 reagent configurations 3-14
selecting chemistry 3-6
troubleshooting analyzed run data 6-2
P troubleshooting chemistry problems 6-5
PCR, general practices 2-7 with SYBR Green I dye 1-6
plus/minus assay
and TaqMan probe-based chemistry 5-3 R
defined 5-2
incorporating an IPC 5-3 radioactive waste, handling xv
instruments 5-2 reagent configurations
internal positive control (IPC) 5-2 allelic configuration assays 4-9
process described 5-3 selecting 3-14
reagent configurations 5-5 selection 4-9
terms described 5-3 reagents, Real-Time PCR control B-9
primer concentrations, defaults 3-22 Real-Time PCR
primer design, guidelines 4-8 defined 1-4
Primer Express software instrument software, ordering B-3
and assay design 3-10 quantification process 3-6
and primer design 4-8 TaqMan detection process 2-3
and small amplicons 3-11 recommendations
ordering B-3 probe concentration 3-25
primer limiting, multiplex assays 3-28 thermal-cycling parameters 3-17
primer matrix relative quantification
and defining limits 3-28 and multiplex PCR 3-45
example of limiting 3-29 calculation methods 3-33
primer optimization matrix, how used 3-22 comparative CT 3-42
comparative CT formula 3-42
primers
and hairpin loops 3-7 relative standard curve
limitations 3-47 example 3-37
multiplex PCR 3-48
probe concentration
performing 3-36
optimizing 3-24
relative values 3-38
recommendations 3-25
relative values, relative standard curve 3-38
probe design, allelic discrimination assay 4-7
reporter dyes, multiplex 3-47
probes, custom products listed 2-4
requirements, dye-binding 2-2
RNA one-step, thermal cycling parameters 3-18
Q RNA quantification
quantification and one-step RT-PCR 3-15
absolute 3-33 thermal-cycling parameters 3-18
calculating absolute 3-34 two-step RT-PCR, and 3-15
calculating relative 3-33 rRNA primers, and multiplex assays 3-28
relative 3-32
RT-PCR
relative formula 3-42 method comparison 1-5
relative method choice 3-33 one-step 1-4
relative vs. absolute 3-32
two-step 1-4
standard curve requirements 3-36
terms defined 3-34 run, outline for performing 3-37
validation 3-42
quantification analysis, terms defined 3-4 S
quantification assay safety

Real-Time PCR Systems Chemistry Guide

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May 11, 2005 12:43 pm, ChemGuideIX.fm

 before operating the instrument xiii Technical Support, contacting ix
biological hazards xvi thermal cycling parameters
chemical xiii allelic discrimination assays 4-10
chemical waste xiv DNA/cDNA quantification 3-17
conventions xii recommendations 3-17
guidelines xiv, xv RNA quantification 3-18
moving/lifting xiii training, obtaining information about ix
sample comparisons, using a calibrator 3-40 troubleshooting
sequence detection system allelic discrimination assays 6-7
software, ordering B-3 allelic discrimination run data 6-8
Services and Support, obtaining ix chemistry problems 6-5
small amplicons, selecting 3-11 DNA concentration effects 6-7
SNP genotyping quantification assays 6-2
assay contents 4-5 two-step RT-PCR
assay information 4-5 primers used 3-6
assay mix 4-5 RNA quantification 3-15
properties of assays 4-5
TaqMan Assays 4-4 U
software
sequence detection system, ordering B-3 UNG, and minimizing carryover 2-6
standard curve Universal Master Mix reagents
absolute quantification 3-53 about SYBR Green I Mix 3-16
about TaqMan master mix 3-15
requirements for quantification 3-36
polymerase benefit 3-16
standardization, and use of endogenous control 3-36
user attention words, defined vii
standards
absolute quantities 3-53
requirements for preparation 3-36 V
SYBR Green I dye validation, quantification 3-42
chemistry described 1-6
optimizing quantification assays 3-23
W
WARNING, description xii
T
waste disposal, guidelines xv
TAMRA dye, and TaqMan probe 2-4
TaqMan Assays
Genotyping and Gene Expression 1-7
product types 1-7
SNP genotyping 4-4
TaqMan exogenous IPC
endpoint assays 5-4
kit design 5-4
TaqMan Gene Expression Assays
description 3-7
TaqMan MGB probes 2-4
allelic discrimination assay 4-7
usage 2-4
when to use 4-7
TaqMan probe-based chemistry
described 1-6, 2-3
plus/minus assays 5-3
specific detection 2-3
used by 1-6
TaqMan probes, types 2-4
Technical Communications
contacting viii
e-mail address viii

Index-4
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May 11, 2005 12:43 pm, ChemGuideIX.fm

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www.appliedbiosystems.com Part Number 4348358 Rev. E