siRNA control (scramble seq) causing cell death - what to try next? (May/02/2010 )
I am trying to knockdown several proteins of interest to see what effect knocking them down has on cell death.
However, my control scramble sequence is causing more death than any of the knockdown siRNA's.
I know it must be the scramble sequence as a transfection control with Lipofectamine 2000 but no siRNA is fine.
I'm not sure what approach to take next as I can't get any meaningful results from my siRNA's until the scramble is not causing any effects. Should I try a different transfection reagent? I have tried two different concentrations of siRNA and get a similar result with both concentrations.
Thanks for any help!
First, have you done a blast search to see if your control siRNA matches to any other sequences in the genome?
Even there is no significant homology with other sequences, it is known that some siRNA sequences could have sequence non-specific cytotoxic or immunostimulating effects. If that is the case you should switch to another control siRNA.
It was a stock sequence siRNA I bought from the company when I bought my siRNA's. I have contacted them, turns out that sometimes the scramble sequence is "toxic" to cells. They are sending me an alternative scramble sequence to try free of charge.