Our lab is trying to design primers for microRNA. Our laboratory wants to test whether the small amount of microRNA that we acquire from the infrapatellar fat pad is enough to run and assay. If you have the information about the minimum amount of microRNA per well that can be detected, that would help a lot too.
Thank you
Erika Lopes
I've designed custom SYBR-green primers using polyA-tailed based cDNA (Ncode) kit. The cDNA kit provides a universal "reverse" primer while your target miRNA is the "forward primer". From my experience, comparing to NGS reads, i was able to amplify miRNAs such as miR-34 and 34c estimated to be 23423 and 681321 reads respectively but encountered high SEM/SD due to high Cts (>29). I would recommend you design primers for miRNAs that are few times more abundant, i've had good success with most custom primers. One caveat to this approach is that you will not detect mature miRNAs, only pre-miRNAs transcript (which i based on capillary electrophoresis sizing).Based on relevant NGS data in the literature (working with the heart), I saw good concordance with pre-miRNA transcript and mature counterpart.
Check your baseline microRNA abundance for some estimation:
http://www.mirbase.org/cgi-bin/browse.pl?org=mmu
You can have a look at Chapter 6 of my PhD thesis for some results and methods.
http://epublications.bond.edu.au/cgi/viewcontent.cgi?article=1195&context=theses
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