placing siRNA seq into miRNA stem-loop - (Aug/15/2011 )
Im having a little difficulty with designing knockdown constructs against a rat gene im studying (stim1). I tried using invitrogens RNAi designer (miR RNAi) but so far all the 6 oligos weve tested have not worked. So i was thinking of trying to place a published siRNA seq that already works into a miRNA stem-loop and see if that would work. My PI prefers to use miRNA rather than shRNA due to higher potency of miRNA and we already have the vectors for it (pcDNA6.2-GW-miR). I was wondering if anyone has any experience doing this, im not sure which stem-loop structure i should use for rats and how to incorporate the siRNA into it. Any advice would be much appreciated. Thanks.
Your RNAi against stm1 gene did not work, was it because the cells are hard to transfect or the siRNA did not work? What do you mean your PI wanted you to use miRNA rather than shRNA? I guess your PI wanted to construct shRNA to knock down the gene, right? I don't think there is special requirement for the loop sequence in rat, and any commonly used loop should work. You can use many online tools to design the shRNA such as ones you can find on Ambion and Invitrogen sites.
the microRNA did not work. i didn't try any siRNA, i used invitrogens online microRNA construct tool for the loop construct and cloned it into one of their vectors they provide thats driven by pol II. Is there any difference between shRNA and microRNA? from what i understand shRNA constructs uses pol III promoters such as H1 and U6, and isnt as potent as microRNA which uses pol II promoter like CMV. thanks
So you actually want to express miRNA, right? In that case, the best way is to clone the precursor miRNA sequence including its ~100 bp flanking sequences on both ends.
ill give that a shot thanks