how do I know if there is a miRNA modulating expression? - identification of miRNA in my system (Dec/03/2009 )
Hi: Could anybody advice me how to trap a miRNA? I believe there is a miRNA modulating my protein, and I would like to get a cheap protocol to determine if that is true. Perhaps a Northern or a RNase protection assay?
Here are a few approaches -- I hope we hear about more!
You can downregulate the miRNA using an antisense or siRNA approach, then look for an increase in the concentration of your protein of interest over time.
You can add additional miRNA to see if you can decrease the concentration of your protein of interest over time.
If you think you know the sequence of the microRNA response element in the mRNA, you can protect that target with a stable steric-blocking antisense oligo and see whether the concentration of your protein of interest increases over time.
Or you can go for a variant on a crosslinking strategy, though that's not an easy assay:
Crosslinking and immunoprecipitation to find targets of miRNA.
Chi SW, Zang JB, Mele A, Darnell RB. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature. 2009 Jun 17.
needmolecularhelp on Dec 3 2009, 04:52 PM said:
OK, thanks a lot. The problem is that I think there must be a miRNA regulaing the system. I donīt know which miRNA is participating, if there is any. Should I knock-down putative miRNA (according to literature) of known sequence?
You will need to have a pretty good idea of which miRNA you want to target. There are programs which locate putative miRNA response elements (MRE, the target sequences) in the 3'-UTR; I've not used these, but based on the comments of folks who have used these packages I am not very confident about their ability to locate real MRE without a lot of false positives. Still, that is a reasonable place to start. Others have suggested using several of these packages and choosing MREs that are selected by all of the packages as the most likely real MREs.
A crosslinking strategy is more likely to lead you to the real MREs, but the procedure will surely take a lot of time to set up and optimize.
In both cases, once you have the MRE you can target the miRNA predicted to regulate by binding that MRE.