Off-target effects of RNAi - (Jul/16/2006 )

Hi, all,

Has any of you noticed/studied off-target effects of siRNA in your research? More than likely we will be uisng shRNA approach for the next step. I appreciate if you could share with me some of the pitfalls that you may have encounted and solutions you have come up with. Thanks in advance.

We had noticed some off-target effects like increased cell death when using shRNA. When we tried to dig deeper in to these effects, we couldn't prove it. Even when we used shRNA invivo (one year), we didnt notice any off-target effects.

Finally, based on some literature serach and our own experiments, we concluded that for invitro experiments, cell types make a difference i.e. certain cell types would had profound off-target effects and some cell types not. Its bcoz of the machinery for such a response is not developed or missing in some cells.

Thanks. scolix. I had very good silencing effects with 27-mr siRNA and we want to further our effort using shRNA with the same sequence. I am concerned that longer shRNA may trigger unwanted side-effects, as reported for siRNA and shRNA >21 mer in length. Have you checked if any sign of IFN activation in vitro and in vivo beside cell growth rate?

Personally, I don't think interferon response is a big concern for off-target effect. We tested many 21-nt siRNA, didn't find any interferon response genes induced. 27-mer is also not longer enough to induce interferon response. We transfected lots of siRNAs which do not have any mRNA targets and observed many of them caused dramatic phenotype changes in cells. The effect could be caused by partial match of siRNAs to other off-target sequences.

We actually tried to look for interferon response but couldnt find any in our cells (hippocampal neurons). We had to get some other cell types (mixed neuronal cultures and microglial cultures) to actually show interferon response with LPS but not with shRNA (21ms).