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timecourse experiment for Western and qPCR - (Nov/22/2013 )

I would like to repeat a timecourse experiment with reverse transfection of siRNA. The knockdown worked very well with the numbers I used but now I have to include a later time point so my cells would be too confluent by then. In general, does anyone know how to do this correctly? Should I transfect more cells and then reseed after 24h, or seed low numbers so I dont have to reseed - but then I can not use these conditions for later experiments because it would be a waste of reagents. Or transfect different amounts of cells for each time point..? For me, best would be to do it like i did last time and then only reseed for the last time point using dome cells from previous time point. Do you think this could work? Thanks so much!


Presumably you are doing these tests to explain some physiological phenomenon you observed at the later time point, so you should handle the cells precisely as you did for the phenotypic experiment.  If instead you are at the phase of optimizing knockdown efficiency at the later time point so that you might see a phenotypic difference later on, then just try every possibility and see what works best.  I personally think that plating very few cells (10% confluency) at the beginning is a good approach, but it all depends on what you are trying to test.


Thanks a lot! I am actually at the stage of optimizing and decided to seed at a low density as you suggested.