Scramble siRNA shows increase in gene expression by Taqman RTPCR - (Oct/07/2007 )
Hi, i've been optimising the siRNA of my GOI for a few weeks now with HiPerfect reagent. (I had tried Lipofectamine but saw a lot of cell death). Everthing was going fine, but I wanted to see a 75-85% knockdown. I tried the experiment again last week and I have achieved the knockdown by using a higher concentration of siRNA. But this time my scramble siRNA showed a two-fold increase in gene expression compared to untransfected control. I haven't see this before. I'm just wondering if anyone else has seen this. If for example I added too much scramble by accident could this be a reason? Does it really matter since I have seen a good silencing of my gene of interest? Thank you.
It's well known that RISC-based knockdowns modulate expression of off-target genes. Even though your scramble sequence doesn't have an explicit intended target, there are plenty of partially-complementary sequences with which to interact.
Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7. Epub 2004 Feb 09.
Expression profiling reveals off-target gene regulation by RNAi. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS. Nat Biotechnol. 2003 Jun;21(6):635-637.