Good night all. I'm using RNAi to knockdown a transcription factor from a primary cell and I need to validate by westernblot. With Ponceau marking i can see that i'm putting the same amount of protein in the wells but every time i got a weaker band of the endogenous control (GAPDH and B-actin) in the silented cell sample. Does anyone have any suggestions to what may be happening?
Dear Lucas,
Of course it can. Especially RNAi with a transcription factor, which can indirectly modulate the expression of different pathways such as housekeeping genes. Another way concerns your control. Are the conditions strictly similar, with a control RNAi ?
Yes, you should check in silico for common regions between the two.
KR
Dear Lucas,
I agree with Gael. It is very likely that you affect housekeeping genes if you target transcription factros. Which one is it anyways? Controls are very important here. You may want to use a transfection only control and a scrambled control to learn about the effects of transfection on your cells. Finally, you may need a different control to proceed with your experiments.
Best
Boris
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