Kuperwasser Lab | qRT-PCR protocol from cell lines to RNA to cDNA to data (PJ Keller)

Collecting pellets

RNA collection from cell lines growing in monolayer culture. Plate cells at appropriate densities and grow to near confluence before collection. Treat cells as needed for individual experiments.

Wash cells with PBS to remove media and serum
Trypsinize with 1-2 ml of 0.05% Trypsin-EDTA solution for 2-5 minutes (or as needed)
Resuspend trypsinized cells with 8-10 ml of media containing serum to inactivate trypsin enzyme, pellet cells by centrifugation at 1000-1200 rpm for 5 minutes
Resuspend pellet in 1 ml of PBS, transfer to 1.7 ml tube
Pellet cells at 3-5000 rpm for 5 min in a refrigerated centrifuge
Pipet off all traces of PBS
Keep pellet on ICE or snap freeze in a MeOH-Dry Ice bath and store at -80C

RNA prep

Prepare RNA from pelleted cells by following the directions in the RNeasy Kit (Qiagen).

-homogenize samples by passing through a 20G needle

-use on-column DNase I digestion to remove genomic DNA contamination

-elute with RNase-free water and keep samples on ice or frozen to limit RNA degradation

-Spec RNA with nanodrop or other spectrophotometer to get A260/A280 ratio and concentration (mg/ml = A260 x 40 x dilution factor if used)

cDNA synthesis

Prepare cDNA by following instructions with the iScript cDNA synthesis kit (Bio-Rad)

Prepare in a PCR tube for each sample (ideally, also include a no RT neg. control):

1 µg RNA

H₂0 to 15 µl (minus the amount for 1 ug RNA)

4 µl of 5X iScript Reaction buffer

1 µl of iScript

20 µl total reaction volume

Run cDNA synthesis reaction on the MyiQ iCycler qPCR machine

5’ at 25C à 30’ at 42C à 5’ at 85C

Dilute reaction 5 fold with 80 µl of nuclease-free H₂0, store at -20 or -80C

qRT-PCR protocol: from cell lines to RNA to cDNA to data

-Prepare enough master mix for your samples + 1 extra

-When setting up the plate, you will need 5 µl of cDNA for each primer set you are using per sample plus an additional well for a negative control (no cDNA)

-One or more of your primers should be a reference gene to use as a control for comparison across samples (i.e. GAPDH, B-actin, cyclophilin, 18s RNA etc.)

-If primers are not well characterized, make sure to include a positive control cDNA as well for that primer

example: 3 samples with 5 primers

Make 5 master mixes (one for each primer) for 4-5 wells (3 samples + one extra for pipetting error or 3 samples + positive control cDNA + one extra for pipetting error). Include additional wells for your reference primer(s) that contain no cDNA, or the no RT control for each sample-in this case the master mix for the reference primer would be for up to 9 wells (3 samples + one positive control cDNA + one no cDNA control + 3 no RT controls + one extra for pipetting error)

Master mix (x N+1 wells with primer):

12.5 µl 2X SYBR green mix

1 µl 2.5 µM Forward Primer

1 µl 2.5 µM Reverse Primer

5.5 µl nuclease free H₂0

20 µl volume

For each well, load 5 µl of cDNA and 20 µl of master mix

Seal plate with sealing film and run on qPCR machine, if necessary, spin plate briefly to move samples to the bottom of the wells and pop air bubbles

qRT-PCR protocol from cell lines to RNA to cDNA to data (PJ Keller)

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