I transfected a cell line with plasmids having shRNA inserts. After transfection, when should I examine the knockdown efficiency? I would like to isolate total RNA and do qPCR for that. I would be grateful for your answers.
Dear Ayça Olcay
As for the GFPknockdown experiment I conducted last week or so, I introduced a plasmid of shGFP into GFP-expressing CHO cells by electroporation, and observed a 90% reduction in GFP intensity by FCM analysis after 48 hours. The 90% reduction in GFP expression persisted for at least 5 days.
I have not evaluated the expression level of RNA, but I assume that it is equivalent to that.
Best,
Hi Ayça Olcay!
Transfection efficiency varies depending on cell type, delivery mechanism, the vector used, and target, etc... Harvesting at 12, 24, and 48 hours is recommended to establish the optimal time point. The best bet is to collect at 48 (RNA) and 72 (Protein) hours post-transfection to determine knockdown efficiency.
Raksha said correct.. But plz check after 24 and 48h. Preferably bly 48h
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