I am trying to knockdown EGFP expression of HEK293 cells that stably express EGFP from Cell Biolabs and not getting any visible knockdown or verified knockdown with RT-PCR. I am using Lipofectamine with an siRNA that I designed and an "old" siRNA against EGFP from Ambion that is predesigned and validated. I transfect 25 to 50 nM of the siRNA and assess for knockdown at 48 hours post-transfection. I've been using a dilution series of total RNA for my RT-PCR analysis (undiluted, 1:10, 1:100 and 1:1000) and 3 ul of each of these dilutions as template. The bands are the correct size but I just don't see any knockdown. I'm wondering if I'm using too much template in the reactions because I seem to get the same results no matter what siRNA or concentration that I try. I'm relatively new to transfection and siRNA work, so any help would be greatly appreciated.