RNA Seq III: Quantitative PCR analysis of mouse retina cDNAs1
Bio 461 Developmental Biology Lab
Saint Louis University
Dr. Judith Ogilvie
*Gloves required
Objectives:

Quantify the expression of genes from cDNA samples run in triplicate using qPCR analysis
Prior to this lab, retinas were isolated from P4 and P6 wt and rd1 mouse retinas. Total RNA was extracted and
converted to cDNAs. Kits are available to perform this conversion using the reverse transcriptase (RT) enzyme
and a mix of poly dT and random hexamer primers. We have two groups of students that have selected genes
based on each of two different RNA Seq expression data sets: Group 1 comparing wt P4 to wt P6 retinas and
Group 2 comparing wt P4 to rd1 P4. We have the following cDNA libraries plus and no cDNA control in a set
of 7 tubes:
Tube #
1
2
3
4
5
6
7
Group 1
P4 wt – rep 1
P4 wt – rep 2
P4 wt – rep 3
P6 wt – rep 1
P6 wt – rep 1
P6 wt – rep 1
no cDNA template control
Group 2
P4 wt – rep 1
P4 wt – rep 2
P4 wt – rep 3
P4 rd1 – rep 1
P4 rd1 – rep 2
P4 rd1 – rep 3
no cDNA template control
Today we will use these cDNA libraries to validate and further study up- or downregulated genes you selected
from the RNA-seq data set.
Careful pipetting is critical! qRT-PCR is a highly sensitive quantitivative technique. Before setting up your
reactions, your instructors will guide you through some tips on pipetting techniques and everyone will practice.
Setting up your reactions and SYBR Green PCR master mix
As a class we’ve designed 26 primer sets to analyze the expression of 24 genes plus 2 housekeeping genes
B-actin and HPRT. PCR reactions will be set up in 96 well plates with each primer set taking up 1 row of 7
wells. The 8th well in each row will be used for control housekeeping genes. Two plates will be shared to run all
of the primer sets. A diagram with the layout of the plates will be provided in class.
 Each student will run 14 PCR reactions: 7 cDNA libraries listed in the table above for each of the two
genes you have selected.
 Each reaction will contain a total volume of 20 uL composed of 2 uL of cDNA library plus 18 uL of PCR
master mix.




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Thoroughly clean bench top and pipettes with ethanol before beginning
Label your designated rows with gene name
Add 2 uL of each cDNA sample to both rows assigned to your two primer sets.
Make sure to orient samples so that cDNA #1 (P4 wt – rep 1) goes into column A and the No
cDNA control goes into column G. Change tips after each loading and use careful pipetting. Give the
plate to the rest of your group mates to load. Then set aside while doing the next step.
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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
Each student will need to make 2 PCR master mixes, one for each primer set, composed of the F
(forward) and R (reverse) primers, 2X SYBR Green reaction mix (contains DNA polymerase, pol buffer,
dNTPs, and SYBR Green), and H2O. NOTE: SYBR Green is light sensitive. Keep it covered with
aluminum foil when possible.

For each primer set make the following PCR master mix on ice:
per sample (uL)
4
10
2
2

master mix
dH2O
2X SYBR mix
Forward Primer
Reverse Primer
X 9* samples (uL)
36
90
18
18
careful pipetting is critical
(keep mixes on ice)
*make extra master mix to allow for
evaporation or tiny errors.
Pipette 18 uL PCR master mix into each of 7 wells for your first primer. Pipette 18 uL PCR master mix
into each of 7 wells for your second primer. Give the plate to the rest of your group mates to load.
Once the plate is fully loaded:



cover plate with an adhesive plate seal making sure all wells are airtight
spin down plate in swinging bucket plate holder rotor centrifuge
load plates into PTC-200 Peltier thermocycler and run “2-step 60C program”*
When the run is finished (~1½ hrs) I will save the data to a jump drive. We will analyze the qPCR data next
week.
*The “2-step 60C program” will run the following thermocycle:
1. 95οC for 30 sec (initial denaturation and heat activation of taq polymerase)
2. 95οC for 5 sec (denaturation)
3. 60οC for 30 sec (primer annealing)
4. 72ο C for 1 min (pol extension)
5. Plate read
6. steps #2-3 39X more times
7. END
Cold Spring Harbor Laboratory, DNA Learning Center, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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