Forty-eight hours after si-RNA transfection, RT-PCR and WB showed that the protein has interfered. Now I want to conduct dosing treatment based on the knockout of the target protein to understand whether the mechanism of action of this drug is related to this base protein.
When silencing is confirmed as here in your case after 48hr, then you can treat the cells with your drug.
Hi,
I'm working with the same experimental design as you. I get nice silencing results from 36 to 96h post transfection. I usually wait for 36h time point before I administer treatment as my protein reacts with overexpression after treatment and is stable until new cell cycle has begun so if any of my protein's RNA is present at the time of the treatment it would overexpress and I wouldn't get the result I wanted.
I would agree to the aforementioned comments. 48 h should work. However, I would test the effects at different time points for drug treatment after 36 - 96 h to get optimal results.
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