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

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