The size of mature miRNAs range between 22 to 26 bp. So, to design primers for the amplification of these sequences by using traditional (general) primer designing protocol is not applicable because PCR methods require a template that is at least twice the length of either of the specific forward or reverse primers, each typically ∼ 20 nt in length. Thus, the target minimum length is ≥ 40 nt, making miRNAs too short for standard RT-qPCR methods.
So stem loop primer is the way to achieve the task and I used several tools for the same including miRNA Primer Design Tool.
In this case forward primer may include mature miRNA itself and may vary, but in most of the cases reverse primer is always found as universal primer, can anyone explain this to me?
I would also like to know if, to design primer for pre-miRNA is not beneficial over mature miRNA sequence?
As you say, the design of these primers is a little different to normal, because you are really constrained by the length of the miRNA.
So the point of the stem-loop primer for the reverse transcription is to increase the specificity by just transcribing RNA molecules with a particular 6mer at the 3' end. That then produces a cDNA containing the full stem-loop primer and full miRNA sequences. The forward primer as you say, just binds to some sequence within the stem-loop primer, so is the same for each assay. For the reverse primer, just make it directly complementary to the rest of the miRNA sequence excluding the 6 nucleotides at the 3' end (so there's no complementarity with the stem-loop primer).
The only problem with this is that the melting temperature of the reverse primer is sometimes a bit low. To get around this, I just added a few extra nucleotides to the 5' end of the reverse primer to increase the melting temperature. The important caveat for this is that for the first few rounds of PCR, these extra nucleotides won't be binding to any template so the PCR will be less efficient initially. For this reason it is important to use a standard curve, rather than deltaCt for quantification. Actually, a standard curve is very cheap and easy to generate just using a DNA oligo with the sequence of the mature miRNA. Somewhat surprisingly, a DNA oligo is reverse transcribed just as well as an RNA oligo so is just as effective in this context.
You can find more details of what we did (including primer sequences in supplementary materials) in our paper here:
Article miR-29a and miR-29b Contribute to Pancreatic -Cell-Specific ...
Thanks @Timothy J Pullen, I will definitely go through your paper. For now I think it is opposite of what you said, reverse primer remain same for each assay while forward primer vary. is it not so?
Because I used several tools and every time I got a set of forward and reverse primers out of which only forward primers vary.
Kindly please correct me if I am wrong.
I am also agree with @Hemant Gupta. I used several tools, which gives different forward primers based on the melting temperature or primer, by adding additional nucleotide sequence, which calculated based on Tm of reverse primer.
Reverse primer remains same ,it is complementary to a sequence within the RT stem loop Primer.
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