Hi everyone,
I am recently performing RNAi by Tet-Inducible shRNA system. Although 2ug/ml doxycline induction for 72 hours could induce the fluorescent tag RFP expression obviously, the knockdown efficiency was very low.
Does anyone can give me some advice?
Thanks a lot!
zhiyuan
When you see RFP expression but not target down-regulation, it is obvious that the shRNA segment is not efficient (either due to inappropriate dicing or inefficient siRNA products). In my opinion, at least 4-5 clones need to be made for a single siRNA target site (right and left orientation) so that few of them will be effective.
Hi Venkatesh，
The siRNA sequences had been tested by Western Blot and it works well. Perhaps there are something else to influence the knockdown efficiency.
Dear Zhiyuan,
The empirical design rules of siRNA (Amarzguioui/Hsieh/Jalag/Reynolds/Ui-Tei) nearly predicts the efficient candidates. However, the same siRNA sequence in the shRNA backbone failed to down-regulate as effective as that of the transfected siRNAs that can be readily processed by RISC. shRNAs are processed by Dicer, resulting various DssiRNAs and eventually, several processed siRNA candidates that are not identical to the initial validated siRNA.
Due to intracellular transcription and processing, there is no standard shRNA design rule (except for the loop, forward and reverse cloning site). This makes it difficult to predict the shRNA's down-regulation efficiency. Hence, we have no option except to do trail and error attempts with 29nt sequence. I mean to say that we cannot expect the same performance of siRNA with shRNA.
In case you come across any influencing factor, please update this thread. I am curious to know.
Hi Venkatesh,
Thanks your resoponse. As you said, we cannot expect the same performance of shRNA with siRNA. And I will update this thread if new advances were developed.
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