Capping shRNA in vitro?

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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Capping shRNA in vitro?

Postby elabrant » Jan 31 2016 2:52 pm


I am new to the forum and hoping to find some clarity. I've seen many protocols that describe in vitro transcription of shRNA's for transfection, etc. However, I have only found a few that describe capping the 5' end of the shRNA. Does capping the shRNA give a marked increase in stability once they're introduced into the cell? I would imagine so, but I need a way to prove (to the powers that be) that it would be worthwhile to add that extra step.

Thank you!
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Re: Capping shRNA in vitro?

Postby nkuperwasser » Feb 13 2016 1:00 pm

In my opinion, it wouldn't be worth the extra step. As I understand it, the capping comes from Pol II based transcripts and its main purpose is for nuclear export and translation initiation (the latter is also linked to the stability of that message). So capping a hairpin RNA which will be digested by Dicer/Drosha etc etc will probably not be that beneficial. Also, Pol-II based shRNAs that arise from mir-based backbones are most likely capped, but the actual silencing part of that RNA will not be capped after the processing, so again, I don't think that capping will be useful.

However, it's a cool idea in itself, but if you introduce capped hairpin RNAs, there may be some other affects (like cap-dependent sequestering of trans factors) that my convolute what you're looking for....

Good luck and sorry if it's not what you were hoping to hear!
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