RNA Seq III: Quantitative PCR analysis of mouse retina cDNAs[1]

Bio 461 Developmental Biology Lab Saint Louis University Dr. Judith Ogilvie

*Gloves required

Objectives:

  • Quantify the expression of genes fromcDNA 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 # / 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
5 / P6 wt – rep 1 / P4 rd1 – rep 2
6 / P6 wt – rep 1 / P4 rd1 – rep 3
7 / nocDNA template control / nocDNAtemplate 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.
  • 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.
  • 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) / master mix / X 9* samples (uL)
4 / dH2O / 36
10 / 2X SYBR mix / 90
2 / Forward Primer / 18
2 / Reverse Primer / 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 Peltierthermocycler 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. 72οC 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 117241

[1] Adapted from a lab developed by Ray Enke at James Madison University