Analyzation of qPCR data to calculate fold change in gene expression using the delta-delta Ct method.

1. Analyzing qPCR data
to calculate fold change in gene expression
using the delta-delta Ct
method
By
Jasleen Saini

2. qPCR (Quantitative PCR) is used to measurethe expression of genes and involves
the useof fluorescentdyes.
The reaction is run in real time and the intensity of the fluorescenceis measured
after each cycle.
The number of cycles required for the fluorescent signalto cross thethreshold is
called the cycle threshold(Ct). Ctvalue is given by the qPCR machine which is
connected to a computer software.
Given below is an example of qPCR data for a control and treated sample:
Sample Reference gene (Ct) Gene of interest (Ct)
Control replicate 1 11.5 28.1
Control replicate 2 12.6 29.2
Control replicate 3 11.8 28.7
Treated replicate 1 11.5 36.3
Treated replicate 2 12.6 35.8
Treated replicate 3 11.8 37.2
Cycle number
Ct = 11
0 5 10 15 20 25 30
Flourescence

3. Step1: Calculate Ct.
Ct is the difference in Ct values for the gene of interest and reference gene for a
given sample. It is done to normalizethe gene of interest to a gene that is not
affected (reference gene).
Ct = Ct (Gene of interest) – Ct (Reference gene)
Sample Reference gene
(Ct)
Gene of interest
(Ct)
Ct
Control replicate 1 11.5 28.1 16.6
Control replicate 2 12.6 29.2 16.6
Control replicate 3 11.8 28.7 16.9
Treated replicate 1 11.5 36.3 24.8
Treated replicate 2 12.6 35.8 23.2
Treated replicate 3 11.8 37.2 25.4
Step2: Next calculate Ct.
Before calculating Ct, average the Ct values of the replicates of control group.
For control, Ctis calculated by subtracting the average Ct value of control
fromthe Ct value of each replicate of control. This is done to normalize the
value of relative expression to 1 for the control.
For treated samples, Ctis calculated by subtracting the average Ct value of
control fromthe Ct value of each replicate of treated samples. This is done for
the relative comparison of treated samples to the control.
Sample Reference gene (Ct) Gene of interest (Ct) Ct Ct
Control rep 1 11.5 28.1 16.6 -0.1
Control rep 2 12.6 29.2 16.6 -0.1
Control rep 3 11.8 28.7 16.9 0.2
Average 16.7
Treated rep 1 11.5 36.3 24.8 8.1
Treated rep 2 12.6 35.8 23.2 6.5
Treated rep 3 11.8 37.2 25.4 8.7

4. Step3: Finally calculate relative quantification(RQ) todetermine the fold
change.
RQ = 2-^Ct
Sample Reference
gene (Ct)
Gene of
interest (Ct)
Ct Ct RQ
Control rep 1 11.5 28.1 16.6 -0.1 1.1
Control rep 2 12.6 29.2 16.6 -0.1 1.1
Control rep 3 11.8 28.7 16.9 0.2 0.9
Average 16.7 1.0
Treated rep 1 11.5 36.3 24.8 8.1 0.004
Treated rep 2 12.6 35.8 23.2 6.5 0.01
Treated rep 3 11.8 37.2 25.4 8.7 0.002
Average 0.005
Averagethe RQ values of control and treated samples to compare the fold change
in gene expression.