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.
Hi Zou,
accomplishing stable transfected cells might help to overcome your issue.
Good luck
Hi Zou, maybe your experiencing a response to your knockdown of your gene of interest. Depending on what your target is, it might be that you have another mechanism that response to the "all of a sudden" low levels of a the particular protein. Check for feedback loops in the litterature as well as check how long your knock down can be kept. Experimentally you need to be sure that you have done enough replicates, meaning that you at least have triplicates for each knockdown setup. What about the total conc. of protein in your samples after siRNA treatment? Do they differ? Hope this gives you some hints. Good Luck!
Hi Zou, I have used siRNA to knockdown target gene protein. In my experiments, its effect can reduced protein level for 4 days. siRNA is not stable off target, so protein level can be up again depend on doubling time of cell type or recover mechanism time of cell after transfection. If you culture cells for long time cell will growth with high %confluence in flask, cells maybe apoptosis and degrade all of protein components, so maybe found protein go down again.
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