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QPCR Data Analysis Assignment - 33

1. The student should not proceed with the qPCR experiment without including a housekeeping gene. A housekeeping gene is important as it acts as an internal control to normalize target gene expression levels and allow for accurate comparison between samples. 2. For qPCR experiments measuring genomic DNA copy number (CN), inclusion of a reference sequence with known constant CN per genome is sufficient for relative analysis between conditions. However, for accurate absolute quantification an external standard curve is needed. 3. While housekeeping genes were historically used as general positive controls for RT-PCR, they are not strictly needed for qPCR experiments measuring only CN from genomic DNA, as long as another reference sequence of known constant CN is included.

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130 views5 pages

QPCR Data Analysis Assignment - 33

1. The student should not proceed with the qPCR experiment without including a housekeeping gene. A housekeeping gene is important as it acts as an internal control to normalize target gene expression levels and allow for accurate comparison between samples. 2. For qPCR experiments measuring genomic DNA copy number (CN), inclusion of a reference sequence with known constant CN per genome is sufficient for relative analysis between conditions. However, for accurate absolute quantification an external standard curve is needed. 3. While housekeeping genes were historically used as general positive controls for RT-PCR, they are not strictly needed for qPCR experiments measuring only CN from genomic DNA, as long as another reference sequence of known constant CN is included.

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shanmuganathan716
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Name : Saran Prabhu.

A
Contact: saranprabhu378@gmail.com
Mobile:8072604237
Title: qPCR Data Analysis.

Date: 04 SEPTEMBER 20XX


Answer three questions and upload them in the portal.

1. Calculate the fold change with raw data given.

In the sheet below, you can find the qPCR raw data results from an experiment. Calculate the
fold change for the following samples using excel and import the calculated data as image here.

HOUSEKEEPING GENE GENE OF INTEREST

R1 R2 R3 R1 R2 R3

CONTROL 17.5 17.4 17.3 18.3 18.4 18.5


(UNTREATED)

TREATED 1 19.1 19.3 19.2 25.5 24.9 25.3

TREATED 2 22.5 22.5 22.6 27.9 28 28.1

TREATED 2 19.3 19.4 19.5 22.6 22.5 22.4


INTERPRETATION:

Treated 1 has more symptoms and more affected than treated 2 and 3 . Treated 2
has less affected and less symptoms.
Treated 3 have not affected and have no symptoms
2. The following picture is a melt curve from a qPCR result that was run. Person “A” is
wondering how to analyze and interpret this. There are three different peaks in this
picture symbolizing 3 different amplifications. What amplification does the first peak
imply? Give a brief reason for your answer. (in about 150 words)

The gold standard for analyzing the products of qPCR continues to be agarose gel
visualization of a portion of the reaction. The presence of a single band indicates a single
product (Figure 2).

uMelt℠: Alternatively, to reduce the number of gels needed to confirm the presence of a
single amplicon, uMelt melting curve prediction software, a reliable, flexible, free online
tool created at the University of Utah, can be used. This program predicts melt curves
and their derivatives for qPCR-length amplicons and is ideally suited to test for multiple
peaks in a single amplicon product.
uMelt can predict the shape of melting curves and the complicated melting transitions
that occur during dissociation. Nearest-neighbor thermodynamics are used recursively
to calculate the helicity of the amplicon at different temperatures through a very
accessible and easy-to-use interface.

uMelt employs algorithms that have been peer-reviewed [1], and users can select from
various design iterations, making the program increasingly popular. The simple interface
comprises a textbox for entering the amplicon sequence, a pull-down menu for selecting
the algorithm version by journal article, and a set of boxes to insert Na+, Mg2+, and
DMSO concentrations (Figure 5).

Although most qPCR master mixes are proprietary, the scientists at University of Utah
have found that the algorithm gives consistent curve shapes under a variety of
conditions and with various master mixes. The main parameter that varies is the
temperature at which the amplicon melts; the shape of the curve and the capacity to
predict the number of peaks remain unchanged.

3. A student at the lab is performing a qPCR experiment, and as she starts her run she
realizes that she forgot to add her housekeeping gene. However, she thinks she should
proceed with the experiment. Is she right, is a housekeeping gene that very important?
Give your perspective on this issue. (in about 500 words)
to measure one other sequence of a constant CN as well. This other gene may be called
a reference or a calibrator.
In contrast, the term "housekeeping gene" denotes a gene that is expressed (transcribed
to mRNA) in any cell(type) at nay developmental stange, and at any time. Such genes
were helpful for RT-PCRs -where mRNAs are reverse transcribed into cDNA which is then
amplified in the PCR- as they could be used as general positive controls. In quantitative
real-time RT-PCR, often housekeeping genes are used as reference genes, what can be
ok but what can also be desastrous. A valid reference gene for quantitative RT-PCR needs
certainly to be expressed, but only in teh cells being analyzed and additionally is needs
to be expressed at constant levels in all experimental conditions you are analyzing. But
this is relevant only for RT-PCR, not for CNA, where you amplify genomic sequences.
So back to CNA: From one sample you measure the Ct of your target gene and also the
Ct of a reference gene (or reference sequence; some genomic sequence with a constant
CN per genome; no need that this is a gene that could be transcribed). The difference in
Ct values (ΔCt) is then comparable between different samples. You can analyze the
mean difference in ΔCt values between your experimental conditions (ΔΔCt) to see if
there is a systematic difference in the CN of the target gene. This would be all that is
required for a relative analysis.
If you want actual CNs, you would need to select a reference with a known CN and you
would need external standards to preform an absolute quantification of the target and
the reference gene.

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