I have adherent cells in a 24 well plate I'd like to incubate with a sample for several time intervals before extracting total RNA for RNA-seq. Is it better to stagger the incubation start times and extract RNA from every well at the same time? Or add sample to all wells at t=0 and, after each time interval is up, pellet cells from certain wells and resuspend in RNAlater?
Hi,
You can collect the cells from the wells you are interested in at the certain time-points. Spin them, aspirate the exceed of the PBS and freeze in LN. Proceed with RNA extraction when you collect all the samples.
Anyway, the best way to isolate RNA from the cultured cells in the well is to add Quizol (Trizol) directly on top of the (washed with PBS) cells. Scratch everything and homogenise by pipetting. Further procedure is routine. The problem of such isolation, is that the cells in the neighbour wells may die in the meanwhile due to various reasons. So the easiest solution would be to incubate a couple of separate plates for different time-points.
Best,
Michail
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