Hi
I am patching CHO transfected cells with Na 1.2 channels. the biggest issue is the efficacy of transients transfection. The protocol sometimes does not show good efficsy and channels expresion is not excellent. This is frustrating when 4-5 or even more patched cells per coverslips does not have current or they are not enought large. Protocol for transfection includes eGFP and lipofectamine and optimem as usual. Does any one have this issue with expressing channels. Or is it normal to check channel expression with Rt PCR or other method to be sure we will have at least more than 60 percent expression. how we evaluate the efficacy of this transfection!?
No, you could check transfection efficacy via using fluorencent-marked controls (e.g GFP-expression plansmids for vectro transfection of Cy5-marked small RNAs) and FACS or microscopic analyses (e.g. see https://www.cellsignal.com/products/sirna/control-sirna-cy5-conjugate/86921).
However, expression efficacy could be checked in mRNA (RT-PCR-based) or protein level (WB, ELISA).
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