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[eesem]

We purchased HEK293 cells from Cell Biolabs that stably express EGFP. The goal was to knockdown the EGFP expression using siRNAs and then rescue it with different chemical compounds. However, I have not been able to get a visible effect of knockdown or show one with an RT-PCR of the mRNA. I'm using 3 uL of total RNA for a series of dilutions (undiluted, 1:10, 1:100 and 1:1000) from the cells to do the RT-PCR verification and now I'm wondering if that is too much and I'm swamping out the effect? I tried to design the siRNAs myself (which may be part of the problem) and generated them with a Silencer siRNA construction kit. Then I found some old validated EGFP siRNA from Ambion that I've tried also with the same results. I've been transfecting the cells with lipofectamine, but I have a sample of RNaiMax that I'm going to try next. I'm assessing knockdown at 48 hours post-transfection and I know that EGFP has a half-life around 24-26 hours. I'm relatively new to transfection and siRNA work so any help would be greatly appreciated.

Thanks,
Erin