How to detect protein-RNA Interaction - (Feb/10/2009 )

Hello,

What would be the best way to determine RNA interaction with my protein of interest? Is there a good protocol for RNA-protein immunoprecipitation? Thanks!

There is a method called RNA-IP. You do IP as usual except that you add RNase inhibitor to protect RNA from RNase. After immunoprecipitation, you do RT and PCR to amplify the pulled down RNA. This paper used RNA-IP
http://mcb.asm.org/cgi/content/full/25/17/7484
also this protocol paper http://www.ncbi.nlm.nih.gov/pubmed/18265380

BottegaVeneta on Feb 10 2009, 09:55 PM said:

A long time ago (well, more or less, but long enough) I used the three-hybrid system. And I remember that it worked very well for that particular RNA-protein combination. But this all depends on the type of interaction you want to test..... it is a lot of work (unless there is something "newer" now). Good luck!

Cheers!

pcrman on Feb 10 2009, 07:18 PM said:

what if we don't know what RNA is being pulled down to design specific primers for it? can you let me know please? thanks.

Wolverena on Feb 10 2009, 10:22 PM said:

Yes, thank you. I was just about to ask.

My protein of interest is a putative RNA binder, and I would like to confirm in vivo.

Curtis on Feb 11 2009, 10:22 AM said:

Mine works wonders with normal random hexamer reverse-transcription. A good antibody, and rather IP lysates with beads already linked to the antibody, because precipitating with the antibody and then binding to the beads does not work well. Afterwards, RNA precipitation in the 99% ethanol step with NaAc and glycogen to maximize yield. It doesn't hurt to put half of the protein fraction on the gel, to check if you actually precipitated your protein. And no urea in the buffer, it killed my precipitation altogether.
I wonder if there's another method to check if the reaction is speficic in vivo. Co-immunodetection in live cells, perhaps?

Atomic force microscopy?

What about an assay to discover which splicing factors bind to certain RNAs? Can you make a bead with a specific RNA on it, then mix a nuclear lysate with it, run a gel, cut the band, mass spec, and find out which proteins bind to your specific RNA of interest?

How does one make the bead with RNA on it? Is there a kit ffor that?

Thanks!