Azure Cielo™ Real-Time PCR Systems

User Manual
Part Numbers AIQ030 and AIQ060
Safety and Regulatory Compliance
Important Safety Information
Please read these instructions before operating the Azure Cielo system.

Electrical Safety Precautions

Be sure to take proper precautions when handling any electrical equipment. NEVER work on any live
circuit, fixture, receptacle, or switch. Safety rules you should follow whenever working with any electrical
appliance include:
• Always shut off power at the main disconnect before changing a fuse.
• Always shut off power to the circuit before repairing or replacing a switch, receptacle, or fixture.
• Always tape over the main switch, empty fuse socket, or circuit breaker on which you are working.
• Always check that the circuit is dead before beginning work. Using a circuit tester or voltmeter can
help determine this.
• Always unplug any appliance before attempting repair.

Protective ground terminal

The ground terminal is located on the inside of the back panel. It is intended to protect against
electric shock in case of a fault.


NOTE: This equipment has been tested and found to comply with the limits for a Class A digital device,
pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against
harmful interference when the equipment is operated in a commercial environment. This equipment
generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the
instruction manual, may cause harmful interference to radio communications.
 peration of this equipment in a residential area is likely to cause harmful interference in which case the user
O
will be required to correct the interference at his own expense.
Changes or modifications not expressly approved by the party responsible for compliance could void the
user’s authority to operate the equipment.

Hot surface warning

U
 nder normal conditions, the temperature of plate is below 50°C and safe to touch. It is
possible that the glass surface temperature will exceed 80°C in rare instances. Please
exercise caution when contact with the plate surface is expected.

CAUTION: No user serviceable parts inside!

For Research Use Only

 his instrument is suitable for research use only. It must be used, therefore, only by personnel who know the
T
health risks associated with the reagents that are normally used with this instrument.

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 Warranty
The Azure Cielo products are warranted against defects in materials and workmanship for one year unless
otherwise outlined on your sales order or www.azurebiosystems.com/warranty. If any defect occurs in the
instrument during this warranty period, Azure Biosystems, Inc. will repair or replace the defective parts at its
discretion without charge. The following defects, however, are specifically excluded:
• Defects caused by improper operation.
• Repair or modification done by anyone other than Azure Biosystems or the company’s
authorized agent.
• Use of spare parts supplied by anyone other than Azure Biosystems.
• Damage caused by accident or misuse.
• Damage caused by disaster.
• Corrosion caused by improper solvents or samples.

Voltage Setting Information

The Azure Cielo system has a power supply that automatically chooses the correct voltage for your country
or region.

CE Conformity
The following Cielo Model Systems, models: Cielo3 and Cielo6 are in conformity with the provisions of the
following EC Directives, including all amendments, and national legislation implementing these directives:
• EN61010-1: 2010
• EN61326-1: 2013
• EN61326-1:2013
• EN301489-1 v2.2.1:2019
• EN301489-17 v3.2.0:2017
• EN300238 v2.1.1:2016
Protection category: IP20 according to IEC 60529

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 Waste Electrical and Electronic Equipment (WEEE)

This symbol indicates that the waste of electrical and electronic equipment must not
be disposed as unsorted municipal waste and must be collected separately. Please
ENG contact an authorized representative of the manufacturer for information concerning
the decommissioning of your equipment.

Ce symbole indique que les déchets relatifs à l’équipement électrique et électronique

ne doivent pas être jetés comme les ordures ménagères non-triées et doivent être
FRA collectés séparément . Contactez un représentant agréé du fabricant pour obtenir des
informations sur la mise au rebut de votre équipement.

Dieses Symbol kennzeichnet elektrische und elektronische Geräte, die nicht mit dem
gewöhnlichen, unsortierten Hausmüll entsorgt werden dürfen, sondern separat behandelt
GER werden müssen. Bitte nehmen Sie Kontakt mit einem autorisierten Beauftragten des
Herstellers auf, um Informationen hinsichtlich der Entsorgung Ihres Gerätes zu erhalten.

Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche

non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere
ITA raccolti separatamente. Per informazioni relative alle modalità di smantellamento delle
apparecchiature fuori uso, contattare un rappresentante autorizzato del fabbricante.

Este símbolo indica que el equipo eléctrico y electrónico no debe tirarse con los
SPA desechos domésticos y debe tratarse por separado. Contacte con el representante local
del fabricante para obtener más información sobre la forma de desechar el equipo.

Denna symbol anger att elektriska och elektroniska utrustningar inte får avyttras
som osorterat hushållsavfall och måste samlas in separat . Var god kontakta
SWE en auktoriserad tillverkarrepresentant för information angående avyttring av
utrustningen.

Contact
Azure Biosystems, Inc.
6747 Sierra Court, Suite A-B • Dublin, CA 94568 • USA
info@azurebiosystems.com • (925) 307-7127 • Fax (925) 905-1816

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Table of Contents
1. Introduction 6
1.1 Azure Cielo Specifications 6
1.2 Plates and Tube Compatibility 7
1.3 Contacting Technical Support 9

2. Installation and Setup 10

2.1 System Placement 10

2.2 System Overview 10
2.3 Connect Azure Cielo Real-Time PCR system to Internet Network 11
2.2 Connect Azure Cielo Real-Time PCR system to PC via Azure Cielo
Manager software 11

3. Device Software Overview 14

3.1 The Home Screen 14
3.2 Experiment Setup 14
3.2.1 Protocol Manager 15
3.2.2 Change Protocol Properties 17
3.2.3 Well Selection Screen 19
3.2.4 Email Feature in Protocol Setting 19
3.2.5 Run Setup 20
3.2.6 Run Protocol 28
3.3 Data Manager 30
3.4 Device Settings 34
3.4.1 General 34
3.4.2 Network 35
3.4.3 Email Server Setup 35
3.4.4 Technical Support 37

4. Frequently Asked Questions 38

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1. Introduction
The Azure Cielo Real-Time PCR instrument is a high-performance tool compatible with a wide variety of
reagents for quantitative PCR. The family of instruments includes the Azure Cielo 3 and the Azure Cielo 6.

1.1—Azure Cielo Specifications

The Azure Cielo instruments have the following specifications.

Azure Cielo 3 Azure Cielo 6

Specifications
Real Time PCR System Real Time PCR System

Part number AIQ030 AIQ060

Product description 96-well Real-Time PCR instrument 96-well Real-Time PCR instrument
with 10.2” touchscreen interface, with 10.2” touchscreen interface,
3 dye channel filters 6 dye channel filters

Sample capacity (wells) 96 96

Reaction volume 5–150µL (10–50 µL recommended) 5–150µL (10–50 µL recommended)

Excitation source LED LED

Detection channels 3 6

Multiplexing Up to 3 targets Up to 6 targets

Thermal element Peltier Peltier

Max. block ramp rate 6°C/sec 6°C/sec

Avg. sample ramp rate 4°C/sec 4°C/sec

Temperature uniformity ±0.2°C ±0.2°C

Temperature accuracy ±0.1°C ±0.1°C

Dye compatibility SYBR Green, EvaGreen, FAM, SYBR Green, EvaGreen, FAM,
VIC, JOE, HEX, CAL Fluor 540, VIC, JOE, HEX, CAL Fluor 540,
CAL Fluor Orange 560, Cy5, LIZ, CAL Fluor Orange 560, ROX,
Mustang Purple TAMRA, TEX615, Quasar 670,
CAL Fluor Red 610, Cy5, LIZ, Mustang
Purple, Cy5.5, Quasar 705

Custom dye/chemistry ü ü
Chemistry capability Fast/Standard Fast/Standard

Detection sensitivity 1 copy 1 copy

Sensitivity Detect differences as small as 1.5-fold in Detect differences as small as 1.5-fold in

target quantities in single plex reactions target quantities in single plex reactions

Connectivity USB, Wi-Fi, Ethernet USB, Wi-Fi, Ethernet

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 1.2—Plates and Tube Compatibility
Azure Cielo Real-Time PCR system is compatible with:
A. Low-profile 96-well Plates
• Low-profile plates have wells of approximate height 15.5 mm (depending on manufacturer).
These modern designs reduce condensate formation and allow for accurate light detection in
fluorescence assays, low-volume reactions, and fast PCR.
• Non-skirted Plate: This plate lacks a surrounding panel. This format fits blocks of most thermal
cyclers and qPCR systems, but may not be suitable for robotic applications without stabilization.
• Semi-skirted Plate: This plate has a short panel around the edge of the plate, providing adequate
support during pipetting and mechanical strength for robotic handling.

*Azure Cielo Real-Time PCR Systems cannot accommodate a fully skirted 96-well plate.
Azure Cielo Systems are compatible with only a non-skirted or semi-skirted 96-well plate*

• Plate color can be White, Frosted or Clear. For assays requiring higher fluorescent sensitivity and
accuracy, white or frosted plates would be recommended.
• Plates should be sealed with Optical Grade Seal Film. Detection of this system is from above and
through the top of each well in the plate. Significant signal loss should be expected if not using
optically clear caps or seals.
• Plate rim (lip): The “rim” of a 96-well plate is a peripheral upper elevation around the plate.
Rimmed plates are NOT compatible with the Cielo system.

• Plate Notch: The notch is a modified corner of a 96-well plate. The Azure Cielo Systems are
versatile and can accommodate notching at any corner position. (A1,H1, A12 and H12)

*The Azure Cielo Systems are not compatible with any 384-well plate*

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 B. Low-profile Tubes
• Tubes are an ideal choice for qPCR experiments of low to medium throughputs. Individual and
tube strips are two most common formats available. Tube strips are available in 8 or 12 well type.
Cielo systems are compatible with 0.1mL and 0.2mL tubes.
• Color of the tube(s) can be White, Frosted or Clear. For assays requiring higher fluorescent
sensitivity and accuracy, white or frosted tubes would be recommended.
• The tubes should be sealed with Optical Grade Flat Caps or seals. Detection of this system is from
above and through the top of each tube. Significant signal loss should be expected if not using
optically clear caps or seals.
• Recommended tube cap features should include a tight fit in addition to optical clarity. Cap strips
are the most common way to seal tubes and plates. Azure Cielo Real-Time PCR systems are
compatible with flat caps, that are optical clear or ultra-clear.

*The Azure Cielo Systems are not compatible with domed caps for tubes or plates*

C. Only a few samples

While the Cielo Real-time PCR system is designed for 96-well plates, many times researchers do not
need to run ninety-six samples. It is common to run only a few samples at a time, especially when
new assays or tests are being evaluated and optimized for best performance.
The lid and detection manifold are very well designed for even heating and detection over the 96-well
landscape. When only running a small number of samples, it is possible uneven pressure over these
few tubes due to the uneven settling of the lid manifold, might damage the strip or single tube
samples during the run.
The easy remedy to this is to evenly distribute samples or strip tubes to different sections of the
96-well plate area. Empty tubes can be used as placeholders at the edges of the 96-well plate area to
allow even pressure from the lid as the run progresses.

Figure 1. Orange circles represent samples to be run. Blue

circles represent empty tubes used as placeholders to ensure the
lid manifold applies even pressure over the entire 96-well area.

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 Figure 2. Filled orange circles show locations of samples across the 96-well format. There are a total of six
samples in tubes. Panel A shows an uneven distribution which may result in deformed or altered tubes at the
completion of the run. Panel B shows a more even and recommended distribution of the same six samples using
two blanks (blue filled circles).

1.3—Contacting Technical Support

For questions about installation, setup, or general use of the system, please contact
support@azurebiosystems.com or call (925) 307-7127, 9am–4pm Pacific time.

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2. Installation and Setup
2.1—System Placement
WARNING: Excessive Weight Hazard – Please use two or more people to lift the system. Failure to do so
can result in system damage and personal injury.
As with all electrical instruments, the Azure Cielo should be located away from water, solvents, or corrosive
materials, on a flat and stable surface with adequate clearance on all sides. The system must remain
stationary during operation.
The system should be placed away from interfering electrical signals and magnetic fields. If possible, a
dedicated electrical outlet should be used to eliminate electrical interference from other instrumentation in
your laboratory.
The Azure Cielo should be installed at no more than 3000 meters above sea level.

2.2—System Overview

Touch screen panel

Start button with indicator LED

USB Type A port

Open button Door

Figure 3. Front view of the Azure Cielo Real-Time PCR systems.

Power Switch
USB Type A port
Power Input
Ethernet port

USB Type B port

Figure 4. Side view of the Azure Cielo Real-Time PCR systems. Figure 5. Back view of the Azure Cielo Real-Time PCR systems.

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 2.3—Connect Azure Cielo Real-Time PCR system to Internet Network
A. Wi-Fi
1. Go to the DEVICE SETTINGS > NETWORK > WIFI on the left edge of the screen.
2. Make sure that the Wi-Fi is on with a strong, available signal.
3. Locate your network from the available networks.
4. Input your password and encryption type.
5. The system should now be connected to the Wi-Fi network.
B. Ethernet
1. Connect a LAN or ethernet cable to the Ethernet port at the rear of Azure Cielo Real-Time
PCR system.
2. Go to DEVICE SETTINGS > NETWORK > Ethernet to make sure connection is properly established.
Note: Depending on your institution’s network security settings, you may need to provide the Mac
Address shown on your Cielo 6 to your IT department to add it into the whitelist of your internet network.
You can find the Mac Address from DEVICE SETTINGS > NETWORK > Wi-Fi > CONNECTION INFO.

2.4—Connect Azure Cielo Real-Time PCR system to PC via Azure Cielo Manager software
A. Internet connection
1. Please make sure both Azure Cielo Real-Time PCR system and the PC are connected to the same
internet network (please refer to Section 2.3).
2. Install Azure Cielo Manager software on the PC with administrative privileges.
3. Use the startup wizard and the SEARCH DEVICES button to locate available Azure Cielo
Real-Time PCR systems within the Wi-Fi network. Select the instrument to be used, and then
CONNECT. The designated instrument and the PC should now connect, enabling run monitoring
and data download at completion of the cycling.
4. If no system is listed to proceed with connection, verify the Azure Cielo Real Time PCR system and
the PC are connected to the same Wi-Fi network.
B. USB connection
1. Connect Azure Cielo Real-Time PCR system to a PC by using USB cable to the standard USB Port
on the back of the instrument.
2. Install Azure Cielo Manager software on the PC with administrative privileges.
3. On the startup wizard, click SEARCH DEVICES button to search available Azure Cielo Real-Time
PCR system, then click CONNECT to connect selected system to the PC.

2.5—Updating the Instrument Software

A. USB format requirements
Most USB drives available will already contain formatting compatible for the Cielo Instrument
Software Update. The USB should be formatted using FAT or FAT32 and if it is not formatted using
one of these file systems, it should be reformatted prior to attempting an update.
To determine the file system in use on the USB, simply plug it into a personal computer using some
version of Windows. Using File Explorer or This PC from the desktop, select the USB drive in question
by double-clicking. It is also possible to hover over the drive, and right click to get a list of menu items.

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 Figure 6. By hovering over the flash drive from “This PC” screen
and right clicking, a menu box will appear, providing numerous
options. Selecting Properties will provide information on the type
of file system in place on the USB. Format will also provide that
information and allow reformatting to proceed if FAT or FAT32 is not
the file system in place.

The file system used has to do with the overall size of the USB drive. One gigabyte or smaller will
likely employ FAT, with larger drives using FAT32. If reformatting the drive, FAT32 should be selected
regardless of size.
WARNING: Please note, reformatting a USB drive will overwrite files already on the drive. If files on

the drive are important, it will be necessary to retain them elsewhere prior to initiating a reformatting.
All files and information will be lost during the reformatting process.
B. Cielo Instrument Software Update Procedure
• Once an appropriately formatted USB drive is available, transfer the Cielo update file to the drive
in the main directory. Do not place the file in a sub folder, nor should there be any other “.bin” files
on the USB.
• The Cielo update file will be named “update####.bin”, with the #### indicating a software version
number.
• Verify that the Cielo instrument has been turned off using the power button on the back.
• Plug in the USB drive containing the software update file into the front USB port.
• Switch on the instrument using the power switch on the back. Wait until the front power button
light shows green. Once green is visible on the front, press and hold the front power button for
eight seconds. Initially the system will beep one time but continue to hold the power button until
there is continuous beeping once per second.
• If you do not continuously hold the power button on the front long enough, it is possible that the
instrument will continue through the process that follows, but please note, that the software will
not load properly. The front power button must be held for the eight seconds to ensure the update
processes successfully.
• A screen on the instrument will display that an update is available, and that firmware has been
located. Select OK to continue with the update to the firmware.

Figure 7. The system will prompt the user that an

update file has been detected. Select OK to continue
with the update.

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 • The Cielo instrument will now begin updating. Do not leave the instrument unattended during this
process, which will only take approximately one minute.

Figure 8. As the Instrument Software Update proceeds, updates to each module will show progress with a moving bar.

• Once the instrument indicates that the update is complete, the USB drive should immediately be
removed from the USB port on the front.

Figure 9. Once the instrument has successfully

updated the software, it will instruct the user to
remove the USB drive and select OK to reboot the
system.

• If the USB drive is not removed, the system will recognize a firmware update, and begin again the
already completed process. If this should happen, just select CANCEL, and remove the USB drive.
Switch the instrument off using the back power button, wait thirty seconds, and then switch back
on the power button.
• The system will automatically restart, with the new firmware in place.

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3. Device Software Overview
The Azure Cielo Icon based software allows you to program and run protocols with a simple user interface.
Protocols and run parameters may be entered using a PC connected via Wi-Fi, USB or ethernet. No PC is
required however, as the Home Screen on the instrument will allow the user this same ability.

3.1—The Home Screen

Figure 10. The Home Screen.

Button Button Name Function

Set up a new run or running an existing protocol –

Experiment Setup
see Section 3.2

Data Manager Access run data – see Section 3.3

Device Settings Access device settings – see Section 3.4

Connection Bar Status and strength of internet connection

Table 1. Buttons in the home screen.

3.2—Experiment Setup
To run a quantitative PCR protocol:
1. ADD (create) a new user or protocol (see more details in the next section).
2. Select existing user and protocol in PROTOCOL MANAGER screen
• If changes for the selected protocol are needed, use the NEXT button to go to
the PROPERTIES screen to edit protocol properties.
• Using the NEXT button , select RUN SETUP to edit the run conditions.
• Select PLAY to begin the run. Information will be displayed in real time on the
screen as the run progresses.

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 3.2.1—Protocol Manager
From the Protocol Manager screen, simply select a listed protocol. While selected, a preview of the
conditions set under that protocol will appear in a separate window to the right of the protocol
manager window.

Figure 11. PROTOCOL MANAGER screen under Figure 12. PREVIEW screen of a selected protocol in
Experiment Setup. PROTOCOL MANAGER.

Button Button Name Function

Home Back to Home Screen

Connection Bar Status and strength of internet connection

Sort Sort in ascending or descending order

• Add User
Add
• Add Protocol

• Delete User
Delete
• Delete Protocol

Go to the PROPERTIES screen to edit the protocol properties

Next
after selecting an existing or newly created protocol

Table 2. Icon buttons in the PROTOCOL MANAGER screen.

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 A. Use the UP button next to Users or Protocol and Date created to sort users and/or
experiments in ascending or descending order.
B. Add New User:
• Tap button to add a new user.
• Tap on ‘Add User’ to add a new user. Enter the new user name using the
system keyboard.
• You can see the new user added to the PROTOCOL MANAGER screen.

Figure 13. Add New User

process flow.

C. Add New Protocol:

• Tap symbol to add a new protocol.
• Tap on ‘Add Protocol’ to add a new user. Enter the name for the protoocol using the
system keyboard.
• You can see the new protocol added to the PROTOCOL MANAGER screen. The PREVIEW
screen of a new protocol displays the default experiment settings.

Figure 14. Add New

Protocol process flow.

Figure 15. The PREVIEW screen of a new

protocol displays the default experiment settings

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 D. Delete existing user or protocol.
• Select the User or Protocol to be deleted.
• While the User or Protocol remains highlighted, use the trash icon to delete the
selected User or Protocol.
Note: Deleting user will delete all existing protocols and data stored under that user.
• The system will prompt you to confirm the deletion. Tap OK to delete or CANCEL to abort.

Figure 16. Delete exisiting user or

protocol in PROTOCOL MANAGER.

3.2.2—Change Protocol Properties

1. Using the PROTOCOL MANAGER, select the protocol to edit properties.
2. Tap NEXT icon to edit the selected protocol.

Figure 17. Select a protocol in PROTOCOL MANAGER.

Figure 18. PROPERTIES screen of a selected protocol.

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 Button Button Name Function

Home Back to Home Screen

Connection Bar Status and strength of internet connection

Tap the rectangular text box to assign a name (or rename)

Protocol Name
the selected protocol

• S elect reaction volume

(default is 20 µL)
Reaction Volume
• Select OTHER* button to customize
reaction volume

Lid Temperature Select lid temperature (default setting is 105ºC)

Off lid heating Turn off lid heating

Light Source Select light source(s)

Back Back to PROTOCOL MANAGER screen

Next Go to RUN SETUP screen

Send run completed notification with result

Email
(.AZD file) attached

Go to WELL SELECTION screen to select wells from

Well Selection
a 96 well PCR plate

*Selected button will turn green from default blue color.

Table 3. Icons and buttons in PROPERTIES screen of a selected protocol.

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 3.2.3—Well Selection Screen
• In the WELL SELECTION menu, a 96-well plate layout will be visible. The Azure Cielo
Real-Time PCR Instrument detects 16 wells or two columns at a time. The default setting
is to collect data from all wells across the entire plate which takes about 9 seconds. If the
experimental plate is not 96-wells, but only a few columns, the overall run time can be
reduced by opting out of detection for unused wells on the plate. The minimum selection
is two rows (or 16-wells). If opting to turn off columns to reduce run time, please ensure
detection remains on for any columns where samples will be placed. Wells that are
considered active for detection are shaded blue and will appear black or empty if turned off
(see images below).
• To deselect wells, tap on the well to clear selection. Wells are deselected in sets of two
columns. Press OK to save changes on to the plate and return to PROPERTIES screen.
Example: Deselected Column 1, 2 and 11, 12.
Note: 2 columns of wells are deselected per tap.
• There is also a CLEAR ALL which will turn off all wells. The user will then select the blocks of
two columns that will be required for detection. Press OK to save changes on the plate and
return to PROPERTIES screen.
• Tap NEXT to proceed to RUN SETUP screen.

Figure 19. WELL SELECTION screen. Figure 20. Deselected columns 1,2 and 11,12 in
WELL SELECTION screen.

Figure 21. Select only Column 11 and 12 after “CLEAR ALL” selection.

3.2.4—Email Feature in Protocol Setting

Important: Make sure that:
1. Cielo device is successfully connected to the Internet. To connect the Cielo Device to the
internet, please refer Section 2.3 (Connect Azure Cielo Real-Time PCR system to internet).

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 2. Email server is successfully setup. To set up email server, please refer Section 3.4.3
(Email Server Setup)
• A pop-up screen with an empty box will appear and will allow user to input an
email address.
• Once the email address is entered, hit OK to continue.
• Proceed with Experiment Setup and run protocol.
• Email notification with results attached will be sent when the protocol run is
successfully completed.
Figure 22. Email ID pop-up screen.

3.2.5—Run Setup

Figure 23. RUN SETUP screen.

Button Button Name Function

Home Back to Home Screen

Connection Bar Status and strength of internet connection

• T urn on Melt Curve by tapping the switch button, then

select the temperature resolution
Melt Curve On/Off
• When BLUE, the Melt Curve is ON; When GRAY, the Melt
curve is OFF

Melt Curve Melt Curve Advance Settings: starting temperature, ending

Advance Setting temperature, resolution, and scan function

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 Button Button Name Function

Temperature Tap to customize reaction temperature

Time Tap to customize time in the step

Tap to assign total cycle number and the step number

Cycle Setting to repeat every time after the last step of the cycle
(see also Go To Module)

Add a step This will insert a step after the highlighted step

Delete a step This will delete the highlighted step

Send the program to a cycling parameter in a previous part

Go To module
of the protocol

Save This will save the Run settings

Camera and/or
Advance settings on camera and/or gradient PCR
Gradient PCR

Back Back to PROPERTIES screen

Run Run the protocol

Table 4. Icons and buttons in RUN SETUP screen of a selected protocol.

1. Temperature settings:
• Tap on the temperature display button for each cycle to change the temperature (°C).
Enter the numerical digits [Example: 60] uusing the key pad on the screen. Press DONE
to save.
• Tap C to clear the entered numerical digits.

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 2. Time settings:
• Tap on the time display button for each cycle to alter the time spent at that step. Time can
be entered in either minutes or seconds. Enter the time using the key pad on the screen.
Example: To set the time to 2 minutes, select Min on the screen and enter the number 2

using the key pad on the screen. Press DONE to save.
Example 2: To set time to 1 minutes 30 seconds, tap on Min on the screen and enter the

number 1. Tap on Sec and enter the number 30 using the key pad on the screen. Press
DONE to save.
• Tap C to clear the entered numerical digits.

3. Go To module and Cycle Settings:

The Go To module and the Cycle Settings are closely linked. Regardless of which step is
highlighted, by tapping on the GO TO button, a two-step set of conditions will be added to the
end of the run (but before the melt curve portion of the protocol). The first step is a 95ºC for 10
seconds followed by a 60ºC for 10 seconds. A repeat cycling default of 30 cycles will also be
visible in a small purple oval with an arrow indicating to which step of the cycle it will go after
completion of the last step. All parameters are adjustable and can be changed. Additional
steps may also be added if something other than a two-step protocol is required
• Tapping the GO TO option will insert two additional steps at the end of the protocol. The
cycles will be repeated for a default of 40 times. User can freely add or delete steps with
the and buttons and adjust temperature and time for these new steps
• Tap on the purple display button at the end of the CYCLING portion of the RUN
SETUP display. The number listed in this purple oval is how many times the conditions
will be repeated. By tapping on the purple display, the number of repeats (cycles) and the
GO TO location can be adjusted (GO TO which step). Be sure to tap the MODIFY button to
save the changes.
• The default setting is for two-step cycling at 95ºC and 60ºC. The default experiment is set
with camera ON (to detect) at the end of the 60ºC step. Please note, if steps are deleted
or added, the default camera setting is OFF. Please review the camera settings to ensure
detection is ON and is happening at the appropriate step.
Example: To set 40 cycles consisting of step 2 and 3, tap on the purple button that

displays number of cycles. A dialogue box will open, and the number of cycles can be
adjusted as well as the step to GO TO.

Figure 24. Go To module on the

RUN SETUP screen.

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 4. Add a step:
• In the CYCLING display, highlight the step after which a step needs to be inserted.
• Tap the ADD menu button (located below the cycling display) to insert a new step. The
default step will be 95ºC for 10 seconds but can be adjusted as described in the previous
section.
5. Delete a step:
• Under the CYCLING display, highlight the step which needs to be removed from the
conditions.
• Tap the DELETE menu button (located below the cycling display) to delete the
highlighted and selected step.
6. Advanced Settings:
A. Activating the Camera:
• The default setting for the camera is ON for the extension step of the default PCR
conditions. As steps are added or altered, users should double check this setting to
verify the camera setting is set to ON and will collect data at any required step during
the cycling. The camera icon will appear light gray when inactive, or dark gray when
active. Camera settings can be altered in one of two ways:
• From the Thermal Profile menu, under the Cycling tab, activate the camera by
tapping on the camera icon within the step detection should occur. The camera
icon will turn dark gray when the camera is set to detect at that step. To turn
detection off again, just tap the camera icon again, and the icon will return to the
inactive state (light gray) .
• Alternatively, within the step where detection needs to be active, tap on the
settings icon . A dialog box will open with a toggle switch that will turn on the
camera. To activate the camera for that PCR, tap the toggle switch and accept
the changes by selecting OKAY or CANCEL to return to the RUN SETUP
screen without saving changes. Double check that the camera is dark gray and
will collect data at the required step.
• The toggle switch in the settings menu will indicate the camera has been switched on
by showing blue behind the button. If the toggle is tapped again, the camera will be
switched off, and will appear gray behind the button.
• For most detection events, the default settings of 50 msec will provide sufficient
exposure from the fluorescent dye in use for data collection. There may be rare
instances when adjustment of the exposure time will be necessary to produce a better
data piece. If an uncommon dye is in use, it is possible 50 msec will be too long of an
exposure, and the signal produced at 50 msec will be over saturated and distorted. In
that case, the exposure times would need to be adjusted down to a shorter exposure
time. If an uncommon dye is not producing sufficient signal during data acquisition,
the exposure time could be adjusted up. The exposure times can be altered from
5–500 msec.
• To adjust the exposure time, select the gears icon from the Run Setup page.

Azure Cielo Real-Time PCR Systems User Manual Page 23

 • Once the Advanced Settings menu appears, select which channel to adjust,
and enter a value in msec (from 5–500). Once a value has been entered, click on
DONE. Be sure to complete the change by clicking the MODIFY button to retain
the new exposure setting or CANCEL to abort it.

Figure 25. Select channel to be adjusted.

• The switch button turns BLUE when the camera is ON and GRAY when the camera
is OFF.

Figure 26. Advance settings screen for

camera and/or gradient PCR.

• The camera at a particular step is active if the icon display is DARK GRAY.
Example: In the image below, the camera is activated at STEP 3 during amplification,
 
and again during the melt curve.

Azure Cielo Real-Time PCR Systems User Manual Page 24

 B. Setting and using the Gradient option:
• A gradient can be an extremely useful option, allowing the user to test several
different temperature conditions within a single run. The Cielo Real-Time PCR
system uses a gradient option that offers different temperatures by columns across
the 96-well plate. The gradient low temperature and high temperature limits are
determined by the user. The instrument software considers those limits, and then
empirically determines the temperature for each column across the plate. Users are
not able to alter those assigned temperatures and a minimum difference of 1 degree
is required for the temperature range

• The GRADIENT option can be accessed by opening the settings within the step to
which the gradient needs to be added. The camera/gradient dialog box will appear.
To enable the gradient option, tap the toggle switch to turn the gradient function ON.
Enter the high and low temperatures that will define the range needed. Please note,
a minimum range of 1 degree is required. When the GRADIENT has been turned ON,
the switch button will turn BLUE. Select MODIFY to save changes. To abort
the changes, select the CANCEL option, which will return the user to the RUN
SETUP screen without saving changes.

Azure Cielo Real-Time PCR Systems User Manual Page 25

 Example: In the following image, gradient is ON for Step 2 and 3 (and OFF for Step 1

and 4). The upper oval shows a range of temperatures, with a series of stacked lines
underneath. Gradient can be turned on for all steps individually.

C. Ramp Rate:
It is possible to adjust the ramp rate for any step in the PCR cycling. Ramp rate is simply
how quickly or slowly the temperature is adjusted up or down, in preparation for the next
step. If an assay is robust enough, or if large numbers of samples need to be processed
over a short amount of time, adjusting to a faster ramp rate could significantly shorten the
overall run time. If a particularly long or high GC content primer is in use, a slower ramp
rate might ensure proper annealing.
• From the Run Set Up Screen with the thermal profile showing, select the small gears
icon to access the additional advanced settings.

Figure 27. From Run Setup, select the gears

icon for the step to be adjusted (red boxes).

• As this advanced settings page is opened, the ramp rate is set to the default setting
of 2º C per second. To alter this ramp rate, click on the toggle button to the right of the
Ramp Rate box.

Figure 28. From the Advanced Settings

window, the Ramp Rate can be adjusted by
switching the toggle button to enter a value
(red box).

Azure Cielo Real-Time PCR Systems User Manual Page 26

 • This will open a new window where an alternate ramp rate between 0.1 and 6.0º C
per second can be entered. Once the desired value has been entered, hit DONE to
return to the Advanced Settings Screen. Finally, hit MODIFY to retain the entered ramp
rate or CANCEL to abort the new setting.
Figure 29. From this menu, enter a new ramp rate for the selected step.

7. Advanced Settings for Melt Curve Analysis:

• Analysis by Melt Curve will need to switched ON using the toggle switch for that option
. The background behind the switch will turn blue when activated.
• Tap the settings icon to access advance setting for the Melt Curve.
• The starting and ending temperatures can be adjusted if the default options are not
acceptable. The default values for the melt curve are from 60–95ºC.
• The resolution of the melt curve can also be adjusted. There are three different levels of
resolutions available. The default level of resolution is set at ºC. When set at 1ºC, this
means for every increase of temperature of 1ºC, the plate will be scanned, and data will
be recorded. Higher resolution means more scans are performed, acquiring more data
with each scan. 0.1 ºC is the highest resolution available with the Cielo Real-Time PCR
system. To save any changes made to the Melt Curve settings, select MODIFY at
the bottom of the window. To close out of the menu without retaining the changes, select
CANCEL .

Figure 30. Melt Curve advance settings screen.

Azure Cielo Real-Time PCR Systems User Manual Page 27

 8. Save Run Setup Settings:
• Save any changes to the protocol by selecting the SAVE icon. This will save all changes
made to the protocol, enabling the user to repeat the same experiment at a later time.

9. Run Experiment:
• To run the experiment, simply select the RUN button once a prepared plate has been
added to the instrument.

Figure 31. The screen display after RUN button

activated. The run time elapsed and remaining
can be monitored using the status box near
the bottom of the run screen. The green meter
indicates how much of the run has been
completed and even provides a percentage
above the status bar. Time remaining is
also provided.

3.2.6—Run Protocol

Figure 32. The screen display after qPCR run finished.

The status box is filled with green, and the percentage
reported is 100%.

Button Button Name Function

Home Back to Home Screen

Connection Bar Status and strength of internet connection

Stop Stop the run

Azure Cielo Real-Time PCR Systems User Manual Page 28

 Button Button Name Function

Pause Pause the run

Run Start the run or continue the run after pause

View curves View amplification and temperature curves of the run

Export Export data file to USB drive

Go back one screen. Selection of this button will return user

Back
to the RUN screen.

Table 5. Icons and buttons in RUN screen of a selected protocol.

• If at any time the user wishes to stop the experiment, select the STOP icon.
• Selection of the pause icon will pause the experiment. Once paused, the button changes to
a RUN button . Press the RUN button to resume the run.
• The progress bar at the bottom of the screen displays the percentage of experiment
completed and time remaining.
• At the initiation of a run, the instrument will provide a default experiment file name and
a location to store the data. The user can select an alternative experiment file name or
destination folder. Whether retaining the default options or customizing, the instrument will
automatically save the AZD raw data experiment file as selected at the completion of the run.
• LID TEMP and SAMPLE TEMP provide information regarding the lid and sample temperatures
at any point during the run. These can be found to the left of the status bar.
• To view the amplification curve and temperature curve during the PCR run, select the VIEW
CURVES icon from the menu. The following screen will appear. Tap the BACK button to
return to the RUN screen.

Figure 33. Amplification curve and temperature

plot during a run.

Azure Cielo Real-Time PCR Systems User Manual Page 29

 3.3—Data Manager

In the data manager screen you can access your run data for export to a USB.

Figure 34. DATA MANAGER screen.

Button Button Name Function

Home Back to Home Screen

Connection Bar Status and strength of internet connection

Sort Sort in ascending or descending order

• Delete User
Delete • Delete Protocol
• Delete Experiment

Export Export Data

Table 6. Buttons in the DATA MANAGER screen.

Azure Cielo Real-Time PCR Systems User Manual Page 30

 
A. Access run data (Amplification Curve and Temperature Curve)
• By selecting the user, protocol and the data file name, the corresponding Amplification Curve and
Temperature Curve will appear to the right in a PREVIEW window.

Figure 35. The Amplification Curve and

Temperature Curve are accessible on DATA
MANAGER screen upon selecting the data name.

Figure 36. Amplification Curve.

Figure 37. Temperature Curve.

1. Amplification Curve
• To examine the Amplification Curve more closely, click on the amplification curve window
within the preview area. The Amplification Curve data will be shown as seen in Figure 36.
• Color coded buttons to the left of the plot window correspond to all the channels available
within that run file. The user can select individual amplification traces by selecting one specific
channel from the options presented at the left of the plot window.
• Each of the color coded buttons will toggle between showing the raw data plot associated
with that dye channel, and turning off the dye channel to simplify viewing of the raw
data plots.
• To return to the DATA MANAGER screen, close the viewing windows by clicking on the CLOSE
option at the very top right of the plot window.

Azure Cielo Real-Time PCR Systems User Manual Page 31

 2. Temperature Curve
• The temperature curve data can also be examined by clicking on the preview version of that
data from the DATA MANAGER screen. By selecting that data window, a new Temperature
Curve window will open as shown in Figure 37.
• The user can view the run temperatures during each cycle, as well as the temperatures
recorded for the lid throughout the entire run. Again, to simplify viewing, there are color coded
buttons to the left of the plot window which permit graphs to be visible or hidden.
• To return to the DATA MANAGER screen, close the viewing windows by clicking on the CLOSE
option at the very top right of the plot window.


B. Delete User
• From the listed users, select the user name to be deleted.
• Use the TRASH button to delete the selected user.
Note: When deleting any user, please note any associated protocols and data files mapping to

that identified user will also be deleted.
• Select OK to confirm the deletion or CANCEL to abort.

C. Delete Protocol
• Select the protocol under PROTOCOL list.
• Use the TRASH button to delete the selected protocol.
Note: The selected protocol and all associated experimental data files will be deleted.
• Select OK to confirm the deletion or CANCEL to abort.

Azure Cielo Real-Time PCR Systems User Manual Page 32

 D. Delete Experiment
• From the DATA NAME list, select the experiment to be deleted.
• Use the TRASH button to delete the selected experiment.
• Select OK to confirm the deletion or CANCEL to abort.


E. Export Data
• Data can be exported to a USB drive using one of the two ports on the instrument.
• Data can be exported via email.
To email files, please verify the following:
• Cielo device has been successfully connected to the Internet. To connect the Cielo Device to
the internet, please refer Section 2.3 (Connect Azure Cielo Real-Time PCR system to internet).
• The email address has been successfully setup and verified. To set up email please refer
Section 3.4.3 (Email Server Setup).

Figure 38. Export screen.

Azure Cielo Real-Time PCR Systems User Manual Page 33

 3.4—Device Settings

Within the Device Settings, the user can customize various information on the instrument. The user can
also access important information from this menu such as the serial number and when the instrument
was last used.

3.4.1—General
• Device Info – Under this heading, the user can find the following information related to their
Cielo Real Time PCR system:

• The user can locate the factory set serial number.

• The currently designated device name is listed. The device name can be renamed by the
end user at their discretion.

• The current system status is available on this page, and provides information related to
the readiness state of the system. Green in the system status indicates that the device is
operating normally and is ready to run.

• The most recent usage of the system (date and time) is also shown on this screen.

• Don’t forget to SAVE any changes made to this screen.

Figure 39. Device Information located under

GENERAL within DEVICE SETTINGS.

• Date and Time – The user can adjust the date and modify the time to reflect their time zone.

• Don’t forget to SAVE any changes made to this screen..

Figure 40. Date & Time under GENERAL within

DEVICE SETTINGS.

Azure Cielo Real-Time PCR Systems User Manual Page 34

 3.4.2—Network
Within the network setting, the user can connect to the instrument with either Wi-Fi or ethernet.
Current connection information is also shown on this page.

Figure 41. Wi-Fi and Ethernet under NETWORK

within DEVICE SETTINGS.

3.4.3—Email Server Setup

Important: Make sure that the Cielo is successfully connected to the Internet. To connect the Cielo

Device to the internet, please refer Section 2.3 (Connect Azure Cielo Real-Time PCR system to
internet network).
To enable email features on Azure Cielo Real-Time PCR system, Cielo must be connected
to the internet network. To establish email options, initial connection to the email server will
be necessary.
Please follow these instructions to enable the email feature:
1. Go to DEVICE SETTINGS

2. Go to NETWORK and then choose Email

Azure Cielo Real-Time PCR Systems User Manual Page 35

 3. Enter the email settings based on email provider. These settings can be obtained from your
IT Administration.

4. Some examples of common server settings are listed in the table below:

Email Provider IMAP Settings POP Settings SMTP Settings

Server: imap.aol.com Server: pop.aol.com Server: smtp.aol.com

AOL (including
Port: 993 Port: 995 Port: 465
Verizon.net)
Encryption: SSL/TLS Encryption: SSL/TLS Encryption: SSL/TLS

Server: imap.gmail.com Server: pop.gmail.com Server: smtp.gmail.com

Gmail Port: 993 Port: 995 Port: 465
Encryption: SSL/TLS Encryption: SSL/TLS Encryption: SSL/TLS

Server: imap.mail.me.com Server: Server: smtp.mail.me.com

iCloud Port: 993 Port: Port: 587
Encryption: SSL/TLS Encryption: Encryption: STARTTLS

Server: Server: Server:

imap-mail.outlook.com pop-mail.outlook.com smtp-mail.outlook.com
MSN
Port: 993 Port: 995 Port: 587
Encryption: SSL/TLS Encryption: SSL/TLS Encryption: STARTTLS

Server: Server: Server:

outlook.office365.com outlook.office365.com smtp.office365.com
Microsoft 365
Port: 993 Port: 995 Port: 587
Encryption: SSL/TLS Encryption: SSL/TLS Encryption: SSL/TLS

Server: Server: Server:

Outlook.com
outlook.office365.com outlook.office365.com smtp.office365.com
Hotmail.com
Port: 993 Port: 995 Port: 587
Live.com
Encryption: SSL/TLS Encryption: SSL/TLS Encryption: SSL/TLS

Server:
Server: Server:
smtp.mail.yahoo.com
imap.mail.yahoo.com pop.mail.yahoo.com
Yahoo! Port: 465 or 587
Port: 993 Port: 995
Encryption:
Encryption: SSL/TLS Encryption: SSL/TLS
TLS/SSL/STARTTLS

5. Once the required information has been provided, TEST the email function by providing an
active email address related to the information listed.

Azure Cielo Real-Time PCR Systems User Manual Page 36

 6. If you do not receive the test email:
a. Confirm the Cielo Device internet settings. Confirm there is a strong and active
internet signal.
b. Check your Email SMTP settings. Verify information has been entered correctly.
c. Restart the device and try again.

7. It is important follow these instructions in the order provided to enable the Email feature in the
Protocol Setup and Data Manager.

3.4.4—Technical Support
Help is available. This DEVICE SETTINGS page provides contact information for the Azure
technical support team.

Figure 42. Technical support information in

DEVICE SETTINGS screen.

Azure Cielo Real-Time PCR Systems User Manual Page 37

4. Frequently Asked Questions
General questions
How is qPCR different than regular PCR?
Quantitative PCR, using fluorescent detection chemistry, can determine starting quantities of materials if
performed correctly with all necessary controls. It can be monitored as the run progresses with the fluorescent
signal measured and recorded during the exponential phase of PCR, when the reaction is at its highest
efficiency. Conventional PCR is evaluated after the run is complete (also known as End Point detection)
and after which reaction components become limited at different rates and slow the reaction. As a result,
accurate quantitation is not possible.

What is a threshold cycle?

A threshold cycle (Ct) or quantification cycle (Cq) is the cycle at which the fluorescent detection chemistry
reaches a level above background. It is determined by the instrument software and is inversely proportional
to the concentration of the target. If there are many copies of the target sequence present, it will reach the
threshold level earlier in the run and have a lower Ct/Cq. If there is a low amount of target, it will take longer
for the cycling to achieve that threshold level and will have a later (or higher) Ct/Cq.

What is the difference between SYBR green and probe-based qPCR? Why would I use one over the other?
SYBR Green detection chemistry is a nonspecific, double-stranded binding dye. Unbound, it has a very low
fluorescent signal, but once attached to double-stranded DNA, it emits brightly and is easily detected. The
nonspecific nature of SYBR Green (and other nonspecific binding dyes) means it will bind to any double-
stranded DNA fragments in an amplification reaction, including nonspecific targets such as primer dimers. By
running a melt or dissociation curve at the completion of the run, specificity of the fluorescent signal can be
determined with a high level of confidence.
The most common probe-based chemistry is hydrolysis probes. A probe-based assay consists of two primers
along with a probe that will target a sequence located between the two primers. Probe structure does vary,
but generally consists of a fluorescent tag on one end of the probe, with a light quenching molecule on the
other end. When the probe is intact, the quenching molecule will absorb any signal from the fluorophore.
As PCR proceeds, the exonuclease activity of Taq DNA polymerase will cleave the probe. By this action, the
quenching molecule and the fluorescent dye molecules are now separated from one another, allowing a
robust fluorescent signal. With each cycle, more of the probe is cleaved in this process, until detectable signal
is achieved. Probe-based detection is highly sequence specific, so melt curve analysis is not necessary at the
end of the run.
There are many factors that should be considered when picking which detection chemistries to use. SYBR
Green is less expensive, and if many different primer sets are in use, might be the more flexible choice. Probe-
based has a higher cost but is also highly specific, and with the large variety of fluorophores available, allows
for multiplexing of assays.

What exactly is a negative control? Why do I need it?

A negative control for PCR is also known as a no template control. This is a reaction that contains all reaction
components, such as primers, dNTPs and Taq DNA Polymerase, but does not contain any template. It is
usually done in conventional PCR as well, with the main purpose to show lack of contamination during set up.

Azure Cielo Real-Time PCR Systems User Manual Page 38

 How do I know how many replicates I need to run?
Technical replicates are necessary to show how small differences in pipetting or template quantities may
impact analysis of the data. If replicates are consistent and show low variation from one replicate to the next,
fewer replicates will be needed.
Detection of the level of change between a control group versus an experimental group will also need to be
considered when determining an appropriate number of replicates. Larger differences will likely require fewer
replicates, with more minute differences requiring more to ensure statistical significance.
Do I need to do a standard curve if I am not trying to determine copy number or quantity?
Standard curves can provide very useful information concerning the qPCR assay even if copy number or
quantity is not the intended result. Running a standard curve over a large dilution series can clearly define
over which Ct values the assay is reliable. Evaluating the standard curve can also provide information about
the efficiency of the assay, and if optimization will be necessary.

What is a multiplex?
A multiplex reaction simply means more than one assay is run within the same sample volume (well). It is
common in probe-based analysis to see duplex or triplex reactions. The benefit to this is that more than one
gene can be evaluated within one sample aliquot, preserving valuable template.
It is important to thoroughly test the combinations of primers used within a multiplex to ensure they will work
well together, and not cross react. Good design can help minimize interactions of primers for different targets.

Why is a melt curve recommended if I use SYBR Green?

SYBR Green is a nonspecific binding dye. It will bind to any double stranded DNA molecule. If there are primer
dimer interactions, or other off target primer to template binding, SYBR Green will not distinguish between
the desired target and other products.
While adding melt curve analysis to the end of a SYBR Green run will delay the results, it will provide valuable
data as to the specificity of the SYBR Green signal that has been recorded.

If I am trying to look at gene expression, how do I know if I am seeing genomic DNA contribution in
my results?
When performing gene expression analysis, there are a few important elements to consider during planning
to help minimize or eliminate any genomic DNA contribution to your results.
Good primer designs are necessary such that only sequences related to the messenger RNA are amplified.
For Eukaryotic systems, this will involve selection of sequence that represents exon to exon junctions that
would be in messenger RNA only. Genomic DNA would contain introns, which would far exceed the base
pair size of the targeted amplicon and would likely not amplify under the conditions used for gene expression
analysis (short extension time). There are many resources available to assist with primer design. There are
also many designs available which have already been tested and proven to work.
During RNA isolation, it is common to DNase-treat at some point during the extraction, or after, to eliminate
much of the genomic DNA. Depending on the extraction method, and the type of DNase treatment, the
effective removal of the genomic DNA will vary widely.
Finally, running an no Reverse Transcriptase (RT) control when cDNA is manufactured can inform to the level,
if any, of genomic DNA contamination present. This no RT control should be run for every extraction sample
and should be prepared at the same time as the + RT samples. Including a no RT control along with any
cDNA samples in a qPCR run can show how much of the signal is due to genomic DNA.

Azure Cielo Real-Time PCR Systems User Manual Page 39

 What is a primer dimer?
A primer dimer is the term used when primers within a reaction interact with each other. This may be
instead of or in addition to the specific target. Many times, within the no template control samples run with
SYBR Green, it is evident that any signal recorded was due to very small products made when the primers
combined to make a nonspecific artifact-the melt curve will show a broad peak (not sharp) with a low
melting temperature (usually 78-79ºC). On a gel for conventional PCR, primer dimers show up as a diffuse
(fuzzy) band at low molecular weight (usually less than fifty base pairs).
If primer dimers are generated, these negative influences will adversely impact the efficiency of the PCR.
The concentration of the primers in the reaction could be reduced or the annealing temperature could be
increased to eliminate this unwanted result.

For my qPCR set up, how much template would be recommended?

A general rule for genomic templates is 50 nanograms of DNA. It is possible to inhibit PCR by adding too
much template to the reaction, so using as little DNA as possible to still get an answer is the best option.
If plasmid template is utilized, very low quantities are needed. Linearized plasmid will amplify better than
circular. Usually, 1 picogram of plasmid DNA can be successfully amplified.
When performing gene expression analysis, the assay will usually load a specific volume of cDNA and not
necessarily a quantity, with the differences normalized to a reference or endogenous control gene.

What is a quencher, and how do I know which quencher to select?

A quencher is a special dye molecule that is attached to the 3’ end of the probe. There are a few different
ones available. Probe providers can make recommendations on the best quencher to use with the fluorescent
dye molecule chosen for detection.
While the probe is intact, the quencher molecule is adjacent to the 5’ end of the probe, where the detection
fluorophore is attached. The quencher will absorb any signal that the fluorophore emits when the two are
in this proximity. Once the exonuclease activity of Taq DNA polymerase cleaves the probe in a sequence-
specific manner during qPCR cycling, the quencher and the fluorophore are no longer held close together. The
quencher is no longer able to absorb the fluorescence emitted, and signal is released. As PCR progresses,
more sequence specific fluorescent signal is freed from the quencher action until the signal reaches threshold
levels and the software assigns a Ct/Cq. Signal continues to accumulate, but the important level is that of
reaching above the background.

Cielo specific questions

I have used other qPCR instruments in the past and I never had to do a compensation experiment when I
ran different dye combinations. Why is it necessary on the Cielo to do this? What if I don’t do it?
A multiplex assay can be executed, and data analysis performed without a compensation experiment.
There is a real advantage to running a compensation experiment however, which can make the data
produced more accurate. Any time multiple fluorescent dyes are employed within a single run, cross talk
or spectral overlap is possible. These terms just mean that a small amount of fluorescent signal can cross
channels and can contribute to the signal detected in another dye channel. By running a compensation
experiment, coefficients can be determined which will enable the software to basically filter out any signal
within a given channel that is not actually coming from that channel but bleeding in from a different detection
window. Compensation need only be completed one time for a combination of dyes and the coefficients
generated can be applied to all future runs using that same combination.

Azure Cielo Real-Time PCR Systems User Manual Page 40

 Can I compare data that I have obtained from a different instrument to that which I have run on the
Cielo? And if so, how do I do that?
It is generally not permissible to directly compare data generated from two different instruments even if the
assay run is the same. Different instruments can have slight fluctuations in the temperatures within the block,
the ramp rate, threshold determinations, among other potential influential factors.

A calibrator sample could be run on both instruments which would allow some comparisons of data based on
that calibrator sample and its result obtained on both instruments.

Even if data has been generated on two different instruments, the final data analysis results could be
compared. For example, if an investigator was running a standard curve, and then determining a copy
number of a target, that result could be compared to a similar run on a different instrument for reproducibility.

What is the difference between the Ct/Cq calculations when using regression versus threshold-based?
Which one should I use?
The Cielo Instrument Software can determine Ct/Cq using either regression or threshold.

Regression will consider the individual amplification plots and their slopes to determine when that sample
has entered the exponential phase of PCR and the corresponding Ct/Cq. Efficiencies of the reactions can also
be determined without running a standard curve.

Threshold based Ct/Cq uses a threshold setting determined by the software which defines where background
signal ends and true signal emerges.

Azure Cielo Real-Time PCR Systems User Manual Page 41

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