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
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Posted 01 May 2011 - 09:08 AM
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/m...-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.
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.
