transfection of MCF7 cells - (Jul/14/2006 )
I am working with MCF7 cell line. I will transfect these cells with antisense oligonucleotides (20-nt) and siRNAs using Lipofectamine 2000 from Invitrogen. Afterwards I will do total RNA isolation and RT-PCR as further characterization. My question is if there is a difference between waiting for 24 hours and 48 hours after transfection to do RNA isolation. I would appreciate if anyone could give me any information on this. Thanks a lot!
I had a good gene knockdown as indicated with a fairly long half-life reporter protein- EGFP by 24 hr, trasncribed from a very strong promoter (CMV), although I did see by 32hr the knockdown seemed to be peaked. For your host cell RNA which expressed at a low to medium level, I think 24 hr would be good.
In one of my studies, we noticed it took some 48hrs - 72 hrs for the peak effect of the knockdown. So u could try a time course of the knockdown at 24, 48 and 72 hrs.