Dear researchers, i need some cost effective protocols concerning the evaluation of internal controls for qualification of Mi-RNAs, validation of Mi-RNA markers and quantifying Mi-RNAs using RT-PCR. Please assist.
Suggestion of objectives for Mi-RNA is also welcome.
I can tell you that regarding miRNA RT-qPCR, you may not have to buy expensive Taq, LNA or other sort of proprietary probes. I have used the NCode cDNA synthesis kit using some simple primer design rules and simple online bioinformatics. I have had great success with this, although if you are looking for templates that are relatively low in copy numbers you may not get reliable results. In my case i had trouble with miR34s which i found were past >30 Ct, the majority of the miRNAs i assesed were not low copy numbers.
To design the primers I used mirbase to get the mature sequence, then i designed a primer against the mature miRNA sequence using IDT oligo tool. There are some rules about designing this type of primers, see page 14 of the manual (second link below). I also used a SYBR green mixture that was from another company, so you will not be locked into buying everything from the company which some kits require this type of approach as their cDNA synthesis is different. The universal primer provided by the Ncode kit will last reasonably long, you cannot buy this invidually. You will have plenty of forward primer which will be your miRNA primer that is going to cost you a lot less than a probe which you have large quantity of. And for the ones you have trouble detecting, you may try a probe based approach for which you will have money saved for because you used cheap custom primers that worked for a lot of your sequences! I also used the miRNeasy kit by Qiagen.
https://www.fishersci.com/shop/products/invitrogen-ncode-mirna-first-strand-cdna-synthesis-kit-2/p-4921669
https://tools.thermofisher.com/content/sfs/manuals/ncode_firststrand_qRTPCR_man.pdf
http://www.mirbase.org/
https://eu.idtdna.com/calc/analyzer
Thank you so much, any suggested published article of your work ? CAN
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