I would like to treat cells with TNF and identify the microRNA that bind to my mRNA 3' UTR with vs. without treatment via next generation sequencing.
I have been looking for ways to conjugate a 3' UTR sequence to GFP or some other tag that I can immunoprecipitate, then elute the attached microRNAs and analyze by NGS, but I am not finding a streamlined method for this. It would even be nice if I could conjugate my 3' UTR to a bead or something then just incubate with microRNA isolated from treated cells then analyze the difference in what sticks to the 3' UTR.
I could come up with something completely from scratch, but if that wheel has already been invented I would rather just try that first.
Hi Ricci
You cannot IP a protein together to a RNA, at least in the same transcriptional unit. There are two alternatives to your approach:
1.- Ago2 RIP: IP with anti-Ago2 antibodies to IP the RISC complex containing all the miRNAs and all the mRNAs and then do deep-seq. This is a sort of genomic-wide approach.
2.- If you want just to test a transcript, you can fuse your 3'UTR to a MS2 binding sequence. This fusion transcript can be IP by using MS2 protein. In this case you can work only with your transcript and eventually pull-down your regulating miRNAs.
Hope it helps. Let me know if you need further help.
Best. Paco
Hello...
Actually I am also looking for a method to identify miRNAs binding to the 3'UTR of my gene of interest.
@Francisco: I really like the idea to produce a fusion protein and do an IP to get all the miRNAs bind to the mRNA.
Is the interaction of mRNA and miRNA strong enough to get really the miRNAs bind? I would expect that the interaction is too weak...I read a lot about CLIP and CLASH method and would assume that cross linking is necessary to get the bound miRNAs.
Could you please give some further details for IP conditions? I would be really happy to get ideas for this procedure.
Thank you very much in advance for help!
bye, Katharina
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