RNA Seq Lab IV: q-RT-PCR data analysis using CFX Manager 3.11
Bio 461 Developmental Biology Lab
Saint Louis University
Dr. Judith Ogilvie
Objectives:
 Analyze PCR amplification curves from qPCR reactions
 Normalize gene-specific qPCR data to a housekeeping gene
 Compare normalized gene-specific qPCR data to a calibrator condition to calculate fold change in gene
expression
In lab last week we set up qPCR reactions measuring the gene expression of 24 genes plus two housekeeping
genes in triplicate from the following mouse cDNA libraries:
Tube #
Group 1
Group 2
1
P4 wt – rep 1
P4 wt – rep 1
2
P4 wt – rep 2
P4 wt – rep 2
3
P4 wt – rep 3
P4 wt – rep 3
4
P6 wt – rep 1
P4 rd1 – rep 1
P4 rd1 – rep 2
5
P6 wt – rep 2
P4 rd1 – rep 3
6
P6 wt – rep 3
7
no cDNA template control
no cDNA template control
We ran these reactions on a PTC-200 Peltier thermocycler. Today we will analyze gene expression data using
Bio-Rad’s CFX Manager Software.
Gene expression analysis
Bio-Rad’s CFX Manager Software is installed on all of the lab computers. I exported the data files from the
cyclers and moved them to a the desktop of each computer. There you will find the two .tad files, 1 for each
Plate.
Viewing and annotating your data in the CFX Manager Software (*Note: CFX software refers to the Ct
value as Cq or quantification cycle, just to make things a bit more confusing)
After opening CFX Manager, open your data file in the
software. Select the Quantification tab near the top of
the window. This will display the amplification curves
for the entire plate, the undefined plate layout, and a
spreadsheet of the quantification cycle (Cq) values for
each well on the plate. Our 1st task will be to define
some or all of the wells on the plate using a plate
template file.
In the CFX software, go to settings  plate set up 
view/edit. We will use the quick guide and help that
come with the program in order to label your wells.
Double-check to see if your plate layout matches the
setup layout that we used last week. If not, go back
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Adapted from a lab developed by Ray Enke at James Madison University
Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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and make the appropriate changes to your plate layout template.
Select OK in the plate editing window to get back to the data analysis window. Right click any well and select
well labels>>>target names to change the plate IDs to the primer name. Look at 1 primer set at a time by
selecting only the wells on the plate layout corresponding to 1 primer set. Use screen capture to save a copy of
the curves.
Scrolling over individual wells in the plate layout with the cursor will highlight corresponding amplification
curves and spreadsheet values in the other windows (likewise, scrolling over individual curves or spreadsheet
values will also highlight like samples in the other windows). Scroll over any amplification curve and right
click>>>trace styles to color code the entire plate. Assign a novel color to each row to code by cDNA sample
for the entire plate.
Quantitative gene expression analysis
1. Select all of the data on the spreadsheet. Copy and paste into an excel spreadsheet.
2. Copy and paste just the C(t) value of the corresponding house keeping gene. For example: C(t) value
of P4 housekeeping gene should align with the C(t) value of P4 gene of interest (GOI).
3. In the adjacent column, you have to calculate the Delta C(t) value. Delta C(t) value is the difference in
C(t) and housekeeping gene C(t) value. Formula to type in is: =F2-G2. Copy this formula to all the rows
beneath so you can have Delta C(t) value for all your samples.
4. The next step is to pick the sample that will be used for calculating Max C(t) value. Samples that you
will use as your 100% expression will be picked for calculating this. For example you can use P4 as a
100% expression if you want to compare to rd1 samples. The Max C(t) value is calculated by averaging
the Delta C(t) values of the sample that you pick for 100% expression. Formula to type in (just an
example): Average (H2:H4).
5. The next step is to calculate Delta Delta C(t). Subtract Max C(t) value from the Delta C(t) value.
Example formula for that is: =H2-Max C(t). Copy the same formula to all the rows below, so you can
have Delta Delta C(t) for all your samples.
6. The final step is calculating the % Expression. Formula for calculating this is: 100*2^-Delta Delta C(t)
value. Copy the same formula to all the rows below. Notice that the average % expression of the
sample replicates that you picked should always be close to a 100%.
Assignment due before you leave today:
Rough draft of data for your Results section for qPCR lab report. Include the following:
1. Image of color coded amplification curves
2. table of Cq values
3. bar graphs of normalized fold gene expression for your individual genes
Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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