I am trying transfection of SiRNA of COPB2 to pancreatic cell lines in 96 well.
Bxpc-3 is difficult to transfect SiRNA following the manufacture's protocol (lipofectamine 3000). So, I have devised some protocol (extend the transfection duration 4hr→15hr, 3000 reagent ammount 0.15μl→0.3μl/ well, DNA amount 0.1ng→0.15ng /well). But after transfection, almost all cells are died.
Please advise me the technical tips of transfection.
Hello,
An important and simple thing to try is to skip antibiotic when transfecting and/or the serum. In my cells, antibiotic-free condition made the biggest difference in survival but other colleagues have reported the serum or both.
If you have tested this in parallel with the other variables (time, lipofectamine reagent amount and siRNA amount), perhaps is best to change to a different transfection reagent, fugene is one of the best, but I usually use it for bigger DNA transfections, such as plasmids. For siRNAs I've used SAINT_siRNA transfection reagent, with 12h-24hour exposure and very little loss of viability and for me it worked better than lipofectamine 3000.
Ideally, at 40-50% confluency, if it's too high or too low it also interferes with viability and transfection efficiency. In my experience, optimizing using a 96 well plate is a bit tricky, and a larger format might help.
I would also recommend using a positive (like GAPDH) and negative/scramble control, to make sure the transfection efficiency is okay, control for off-target effects and to assess if perhaps the loss of viability is part of the knockdown of your target protein. In the last case, it's also important to then test with a different sequence of siRNA, to better control for off-target effects of targeted sirna.
Hope it helps ;)
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