Hello,
I'm conducting siRNA-mediated gene knockdown experiment, and after 48 hours, I re-transfected again because I will be collecting cells 72 hours after seeding cells.
However, I noticed that compared to cells transfected with siRNA just for 48 hours, samples with 72 hours siRNA transfection showed the opposite of gene knockdown (according to the qPCR data, the gene expression level increased by five folds).
I wanted to know what might have caused this, and if this has anything to do with toxicity of lipofectamine (if there is any). Thank you!
repeat the same experiment with random siRNA. If result will be the same, you may consider toxicity; if not, pick up another oligo. Good luck
For your qPCR primers are they complementary to your dsRNA or a different region of the transcript?
If your qPCR primers are complementary to the dsRNA then you may just be amplifying your dsRNA. You should make sure your qPCR primers are downstream for your siRNA binding site in your target transcript and have no overlapping complementarity to your dsRNA.
Hope that helps.
Typically its OK to re-transfect cells every 2-3 days, if you want to have a prolonged knockdown. However you should be very accurate and use min possible siRNA and transfection reagent concentration. I typically do (1) 2-3 concentrations of siRNA, under optimized transfection conditions (balancing max knockdown with min cell toxicity) (2) several time points (3) many controls
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