I did shRNA knockdown of a protein and did WB. I did not see any protein expression.Result is reproducible. Still I tried different antibody from different company, again i don't see any protein expression. After QPCR or RNA seq of targeted gene expression of sh knockdown cells is very similar to scrambled! which is very strange for me.
any help from any one for this strange result?
Thank you.
Hi Thomas,
I did check the protein expression in scrambled and there is expression.I could see clear band in WB. I always compare scrambled cells vs Sh cells.
Your shRNA might only repress the translation machinery on our mRNA of interest. Therefore, protein synthesis is blocked but mRNA is steady available.
To firm this possibility up, you can study the interaction between the siRNA (which is generated by shRNA processing) and its target site within your mRNA of interest via luciferase reporter assay. If this assay confirms the interaction, then you can conclude that only protein synthesis is repressed.
Hi Andrey,
I agree with you but what would be the mechanism? how to explain for the reviewers!
Hi Hossein,
We also have the same thoughts. But we have to prove by experimentally. Do you know any publication which explained the method which you have mentioned? or any detail methodology?
Thanks.
Hi Nishant,
You first need to clone your mRNA (actually cDNA) into pGL3 or psiCKECK2 plasmids, downstream of luciferase to generate the wild-type construct. Then you need to know which part of your mRNA is targeted by siRNA. Then you also have to mutate that part, to make the mutated construct, and then compare the luciferase activity of both constructs in the presence and absence of shRNA.
If luciferase activity is decreased in wild-type construct in the presence of shRNA, and remains steady in mutated construct, then you can confirm that your shRNA is targeting your mRNA of interest directly. Then, by showing western blot and down-regulating effect of shRNA on the protein you may conclude that the shRNA only represses the translation and does not result in mRNA degradation.
I have attached a paper, you can take a look at the first result for the better understanding.
how to explain for the reviewers!
Once your result in expression via qPCR is confirmed by WB, and no side effect is observed, this should be enough for reviewers. Or if there is any effect displayed by your protein on it's counterparts, you may try measure it as well.
Hi Hossein,
Thanks a lot for the explanation and for research article.
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