P. Keller 8/2011
qRT-PCR protocol: from cell lines to RNA to cDNA to data
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.
1. Wash cells with PBS to remove media and serum
2. Trypsinize with 1-2 ml of 0.05% Trypsin-EDTA solution for 2-5 minutes (or as
needed)
3. 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
4. Resuspend pellet in 1 ml of PBS, transfer to 1.7 ml tube
5. Pellet cells at 3-5000 rpm for 5 min in a refrigerated centrifuge
6. Pipet off all traces of PBS
7. 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  30’ at 42C  5’ at 85C
Dilute reaction 5 fold with 80 µl of nuclease-free H20, store at -20 or -80C
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P. Keller 8/2011
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
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