Hi,
I have established the best transfection condition that maximizes the silencing effect of Dharmacon siRNA (with Dharmafect-1) on my gene of interest (GOI). My study aims to evaluate the effect of switching off the GOI on cell proliferation. I realized, however, that the best cell concentration at which the silencing occurs is incompatible with a cell proliferation assay. Indeed, 48 hours post-transfection (the time when I have about 80% of the GOI silenced) cells are already at the confluence.
So I was wondering if I can detach cells 24 hours after Dhamacon siRNA transfection and reseed them more diluted. By this approach, I could evaluate the effect of GOI silencing on cell proliferation after 3-4 days. Does anybody have experienced this approach?
I have done this before and it worked for me. I transfected cells at 80% confluence and the following day, I reseeded them for two different assays (wound healing and cell proliferation). I then harvested the same cells after each assay for western blotting to confirm knockdown. Dharmacon siRNA is very good in my experience and can maintain knockdown for a number of days. Good luck!
Great Tarryn! thanks a lot for your answer. This makes me very confident that things will work fine.
Gianluca Occhi reseed cells in serum growth medium (cells will not attach to the plate without FBS) and do not overtrypsinize them. Good luck!
Yes you can, but use a gentle cell detachment agent. You might need to seed at a higher density than usual or add some more FBS in case you see problems in cell attachment after the transfection
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