I did a knockdown, after checking the level of protein expression with Western blot, I got some percentage let's say like 60 to 70 % of knockdown, but when i tried checking with qPCR to check the transcription level it was as if there was a complete knockout, what could have been the reasons, I am confused can some help me with an explanation? You help will be highly appreciated.
Hello Innocent Agida
Are you using siRNA? If yes, then you are most likely to face this problem.
You need to consider protein stability which is highly variable. It may so happen that your target protein may be highly stable with a longer half-life. In such a case, you may consider choosing shRNA-mediated gene silencing method over siRNA. While making a choice, you will have to consider the length of the assay as well as the half-life of the target protein.
siRNAs are transiently expressed in cells, while shRNA is stably integrated into the host cell genome. As cells divide, the shRNA is passed on to daughter cells. Using lentiviral vectors for expression of shRNA, provides permanent knockdown without needing to transfect the cells multiple times.
The protein levels subsequently go down over time because the shRNA constantly keep suppressing mRNA.
So, if you are trying to knockdown the expression of protein that has a long half-life, then stable expression of shRNA may be required.
Best.
It is also possible that your gene is transcribed at a very low level but translated at a high level from those transcripts.
@Malcolm Nobre and Amy Klocko thanks for the response, I appreciate!
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