RNAi in combination with adenoviral-mediated over-expression - (May/08/2012 )
I work with hard-to-transfect primary human cells. I need to use adenoviral-mediated transduction to over-express genes of interest in these cells but can get acceptable gene knockdown using standard siRNA (without viral delivery). I want to knockdown expression of one gene in cells I've over-expressed another gene in. My problem is I get massive cell death even with my scrambled siRNA when I do this. I've tried transfecting with the siRNA, waiting 48h then infecting with the adenovirus and I've tried simultaneously introducing the siRNA and virus. (One of the genes I'm over-expressing with the virus causes apoptosis & in theory silencing the other gene should protect against this so it won't really work to do the viral infection first then knockdown the 2nd gene which is why I haven't tried that combination). I can use the same transfection reagent (Xtremegene HP) for both siRNA and virus & my mock transfected cells don't die. Has anyone else encountered this problem? Any suggestions or any ideas why the virus/siRNA combination is toxic?
Hi, I can understand your problem. I have few questions and suggestions for you. Are you sure about Xtremegene HP works better than Oligofectamine for your siRNA transfectoins. Dont you think 48hrs is way too long for post siRNA transfection. Also make sure that how many times you transduce cells after siRNA treatment. It would be best to reduce your overexpression by diluting with media to control your viral titre if cells die soon. Also make sure whether you are using way much of your siRNA volume in case of cell death. I also hope your viral stocks (stable cell line) are fresh. Hope this reply helps. Good luck.