Hi, the gene of my interest has a 3'UTR of nearly 9.8kb. I am planing to perform luciferase assay for studying the miRNA-target interactions. So is it okay if i could clone those regions where miRNA is binding to 3'UTR (acc. to the insilico tools) or should I use any other stratergy for the same. Please suggest if I could study the miRNA-3'UTR interaction through any other assay.
That is a good question. Although the 3'UTR luciferase assay is regarded as one of the gold standards to show that a gene is regulated by a specific microRNA, the test may involve some un-physiological parameters that can make the assay a little bit artificial. Therefore, I would say, since the assay tends to be artificial, it is probably okay to just clone a fragment of the 3'UTR that contains the putative binding sites for the microRNA.
Alternatively, you could use site blockers (for example, http://www.exiqon.com/mirna-target-site-blocker). I have not used this approach but it seems a viable alternative approach, that combined with a 3'UTR luciferase assay of wild-type and mutant 3'UTRs, should convince most readers that the putative target can indeed be a target of your microRNA. Whether it is a relevant target is another question.
Hope this helps.
I have previously used (and published with) shorter synthetically generated oligos to clone into a dual luciferase reporter vector for this purpose. It greatly expedites screening large numbers of potential miRNA binding sites and is much cheaper than cloning multiple target sites conventionally. additionally, it makes point mutation of the predicted binding site for controls trivial to generate (just order another sequence).
This may not be as ideal as cloning a fragment with a large amount of flanking sequence (as we know that flanking sequences can play a role in site availability, etc), but as Thomas pointed out, the system is already very artificial. Additionally, the flanking sequence itself may be a complicating factor as it could potentially harbor a binding site for your miRNA that was not predicted.
Hi Dear
During my Master's, we just clone the predicted 3UTR sites for luceferase assay.
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