I want to study microRNAs. I have about 15 candidate miRs but the concentration of the sample (extracted from blood) is not enough to converse RNA to cDNA by step-loop technique (my protocol lab used 1 ug/each miR), so I design to use the universal reverse transcription to converse RNA to the cDNA and then I will use RT-PCR to investigate microRNA expression. However, I don't want to buy the additional kit because I already have the forward primer and SYBR green reaction kit but I don't have the universal reverse primer. Would you please advise me to find only the universal reverse primer for PCR or I should do another way to converse cDNA such as try to reduce the concentration of the extracted total microRNAs by using stem-loom miRs because I already have the step-loop primer to save the cost or something else?
We have had a lot of success using this protocol for our miRNA RT-qPCR. It's very cheap and gives great amplification:
http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-11-70
Instead of using a universal stem-loop primer, you first poly-adenylate your whole RNA sample and then do your RT with a universal microRNA RT primer containing a poly-T tail. It's analogous to using a typical random hexamer RT primer, but the binding efficiency for microRNAs is greatly increased by the poly-A/poly-T binding. And the great thing is that these are just regular primers, so they cost basically nothing. The paper should include a link to the software you can use to design your own miRNA primers. Hope this helps.
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