cellular death during dsRNA transfection. - (Jan/06/2006 )

Hi, I'm trying to transfect cells with pRetroSuper-puro + Lipofectamin 2000 but I see a lot of cellular death. I've tried to adjust Lipofectamin 2000 amount and cell density, but the problem persists.
Could Interferon Response be the cause of the problem??
Maybe the problem is DEPC-water I used to dilute DNA (DEPC-water made in my own-lab)???

Thanks!!

-yarince-

Hello,

It should not be very hard to troubleshoot the problem.

Does lipofectamine 2000 alone (mock transfection) cause cell death? - If yes, your cells are sensitive to lipo

Does control shRNA cause cell death? - If yes, the vector may be toxic to cells

If the answer is no to both, an interferon response may be the culprit.

-pcrman-

Thank you pcrman for your answer!!!

I'm trying mock transfection next time.

But, as for control shRNA, I havent got this molecule, just specific shRNA for my gene... I thought that cells transfected with specific shRNA which are puromicin resistant but dont knock-down target gene could be my control cells.... is it correct??

Thanks!!!

-yarince-

Every RNAi transfection should include mock and control siRNA or shRNA. If there is no control shRNA (which has no homology with your gene and all other genes), how do you know the effects (whatever they are) caused by your gene specific shRNA are sequence specific?

-pcrman-