Triage of miRNA targets by qPCR - (Apr/30/2011 )

Hey everyone. I need some help figuring out how to proceed with my project.

Using TargetScan and miRanda, I identified 11 microRNAs that regulate the gene we are interested in. We then attempted to cut the list down to hopefully 1-3 target miRNAs by determining which were expressed in the same brain nucleus as our target gene by qPCR using the TaqMan system. I ran 8ng/RT and U6 is the internal control (average Ct=24). Eight animals were tested, with very similar Ct values in all samples.

Five of the miRNA had absolutely no expression (Ct>40, same as NTC) while the remaining six had Ct values between 24-36 (∆Ct values between 0 and 12). Two have good Ct values of 25 and 26, while the remaining have Ct values around or above 30 (∆Ct 6-12).

Since there is a limited budget, I can't test the functionality of all six in a luciferase reporter system. I want to proceed just testing the 2 best miRNA, but a collaborator thinks we should test all of them (or at least most).

What is an acceptable ∆Ct value for proceeding from here? I think that a ∆Ct greater than 6 is to big of a difference for the miRNA to be reasonably expressed in that nucleus. But I also don't want to throw out my actual regulator.

I tried looking through the literature, but I no one has seemed to publish their rational for choosing miRNAs after this stage.

Thanks!

-iMatt

Actually, testing 6 miRNA with reporters isn't that bad - I found that several of the miRNA I was interested in were clustered, so the need was cut down to about half. Combined with the cheapest prices for the reporters here http://abmgood.com/miRNA%28microRNA%29/3%27UTR-miRNA-Reporters.html , it didn't cost nearly as much as I thought it would.

You might be able to work out a bulk deal for the reporters - I fenangled a 20% discount for 5 of them. They actually have a great sale on right now for all miRNA products, so bulk discounts might not work. I stocked up on primers and my next set of reporters last week, but didn't get the extra discount. The sale should still be on.