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Quick How-To: Calculating Relative Expression of Target Genes Using QRT Data

qRT-PCR is a method to quantify gene transcripts in a sample using fluorescent dye and PCR cycling. The amount of transcripts is determined by the cycle threshold which is the number of cycles for fluorescence to reach a threshold. The higher the cycle threshold, the less transcript was originally present. Relative expression between samples can be calculated using the difference in cycle thresholds and accounting for differences in starting cDNA levels using a reference gene.

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34 views

Quick How-To: Calculating Relative Expression of Target Genes Using QRT Data

qRT-PCR is a method to quantify gene transcripts in a sample using fluorescent dye and PCR cycling. The amount of transcripts is determined by the cycle threshold which is the number of cycles for fluorescence to reach a threshold. The higher the cycle threshold, the less transcript was originally present. Relative expression between samples can be calculated using the difference in cycle thresholds and accounting for differences in starting cDNA levels using a reference gene.

Uploaded by

Aditi
Copyright
© © All Rights Reserved
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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qRT-PCR Basics:

qRT-PCR (quantitative reverse-transcriptase PCR), also called qPCR, is a method to quantify the amount
of gene transcripts present in a sample.

For qPCR in the Whyard Lab, we use a PCR mix called SsoFast EvaGreen Supermix (from BioRad). This
mix contains a special, fast cycling Sso7d-fusion polymerase and EvaGreen, a proprietary fluorescent dye
that binds to amplified DNA. Therefore, fluorescence will accumulate in each sample well after each PCR
cycle as more DNA becomes amplified.

When you run a qPCR on the CFX machine, the level of fluorescence in each well is measured after each
cycle. Curves generated from this data show the accumulation of fluorescence in each well over the
course of 40 PCR cycles.

Definitions:

Threshold – The relative fluorescence level where signals from amplification of cDNA are above
background signals. The optimal threshold level is calculated by the CFX computer program. It is usually
the level of fluorescence where exponential phase of amplification starts.

Cycle threshold (CT or Cq) – The number of PCR cycles required for your sample to reach the optimal
fluorescence threshold.

CT values are used to calculate the relative gene transcript levels among your samples.

The higher the CT value, the longer it took for fluorescence to reach the threshold in that sample,
therefore there was less transcript in the sample to start.

Delta CT (ΔCT or dCT) – the difference between two CT values, usually between reference gene and
target gene or control sample and treated sample.

2ΔCT or 2dCT (sometimes written as 2^dCT) - Because PCR products are amplified exponentially (i.e.
number of amplicons double after every cycle), 2 ΔCT gives the fold-change difference in expression
between samples.
Calculating relative expression of target genes using qRT data:
Consider the following data:

Sample Target Ct value

Control S7 18

Treated S7 21

Control ker 20

Treated ker 25

Determine the difference in the level of kermit (ker) expression in your control sample compared to your
treated sample by doing the following calculations:

1. Calculate the dCT of ker between your control sample and your treated samples:

dCT for ker = (Control ker CT)-(treated ker CT) = 20-25 = -5

2. Calculate the fold-change difference in expression of ker between your control and treated samples
by doing the calculation 2dCT:

2dCT for ker = 2-5 = 0.03125

This value means that there is 0.03125x the amount of ker in your treated sample compared to your
control sample. (Conversely, this means that there is 25 = 32x the amount of ker in your control sample,
compared to your treated sample).

From these calculations, you might think there is much less ker in your treated sample than in your
control sample than there actually is. This is because you haven’t taken into account that there was a
different amount of starting cDNA loaded into each well of your qPCR plate.

We use the reference gene (S7) to compare the amount of starting cDNA in each sample.

3. Calculate the dCT of S7 between your control and treated samples:

dCT for S7 = (Control S7 CT)-(treated S7 CT) = 18-21 = -3

4. Calculate the fold-change difference in expression of S7 between your control and treated samples by
doing the calculation 2dCT:

2dCT for S7 = 2-3 = 0.125

This means there was only 0.125x the amount of starting cDNA loaded into your treated sample wells
compared to your control sample wells. (If the dCT for S7 was equal to 0, then 20= 1, meaning there was
no difference in the amount of cDNA loaded into your control and treated sample wells).
5. Determine the ACTUAL difference in ker expression in your treated sample compared to your control
sample by dividing your 2dCT for ker by the 2dCT for S7:

0.03125/0.125 = 0.25

Therefore, there is actually 0.25x the amount of ker in your treated sample compared to your control
sample.

6. To calculate the amount of knockdown of ker expression in your treated sample, you assume the level
of ker expression in your control is equal to 1. Therefore, you can do the following calculation to
determine your percent knockdown:

(1-0.25/1) x 100 = (0.75/1) x 100 = 0.75 x 100 = 75% knockdown of ker after treatment

SUMMARY OF CALCULATION:

2dCT of target gene


2dCT of reference gene

(where dCT = control sample CT-treated sample CT)

You can then calculate % knockdown if desired by using the calculation in step 6 above.

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