I performed a siRNA knockdown in vitro experiment.
Target gene protein expression was decreased at 2 days after knockdown, however, it went back to normal level in compare with vehicle and ssRNA control at 3 days, and go down again at 4 days.
I have performed this experiment 2 times, got very similar results.
I prepare the siRNA in non-antibiotic medium, after adding siRNA for 4hrs, add 2x medium w/ serum, but still w/o antibiotic, change to normal culture medium over night. Keep culturing them till 2, 3, 4 days after transfection, then Western Blot.