We have the sequence of an siRNA we have used to knock down a gene of interest. Rather than keep ordering it, we converted it to shRNA to clone into the pU6YH vector.
Cloning worked, cells are transfected, but no knockdown relative to siRNA control. Can all siRNA's be converted to shRNA? We have siRNA-resistant constructs that we do not want to re-design. Could the sequence/length of the loop make a big difference (we are using one that worked for someone else)?
Not all siRNAs will work as shRNAs. In fact, most of them don't. It is not clearly understood why, but the loop also makes a difference. I would recommend you either order a larger scale (usually more economical, you can get them to ship in smaller aliquots), or try out a few pre-designed/validated shRNAs and use the one that works best instead. Sigma Aldrich has a good collection of validated TRC shRNA clones.
Some commercially available siRNAs are modified (e.g. 2' O-Methyl) to "force" the antisense strand been recruited by the RISC, also many siRNAs may start with C or T as the first nucleotide in the sense strand. However the U6 promoter for shRNA expression prefers G as the starting nucleotide and H1 is more flexible (may prefer A or G). Overall the siRNA to shRNA conversion may not be 100% compatible but doable.
the rules for shRNA are slightly different. I would design shRNAs at the website Rnai codex. The has to be mismatches at the transcribed shRNA that folds over so that the RISC complex can open it during processing. Also Invitrogen has algorithms to convert siRNA to shRNA at their RNAi portal.
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