It appears that your primers may not be specifically amplifying the mature hsa-miR-1587, despite the clean melt curve and normal amplification profile. The observed band size of 120–150 bp on the gel suggests that the primers might be binding to precursor or primary miRNA sequences rather than the mature 22-nt target, which should yield an amplicon of approximately 50–60 bp when using stem-loop qPCR. This often happens when the forward primer does not align precisely with the mature miRNA sequence or is too long, as in your case, where the 20-nt forward primer might be extending into upstream regions. To resolve this, ensure the forward primer perfectly matches only the mature miRNA sequence (typically 18–20 nt in length), and confirm that the reverse primer binds exclusively to the stem-loop sequence without extending into the template. Additionally, align your primers against the mature miRNA and precursor sequences using tools like NCBI BLAST or miRBase to verify specificity. Redesigning your primers with strict adherence to mature miRNA boundaries and validating the binding sites in silico should help generate the expected shorter amplicon and improve result specificity.

Hi Sima,

I checked you assay - however I have a question. Which kind of DNA a are you using for your assay? Do you isolate total RNA or which kit do you use for furthe isolation of miRNA?

When I checked you assay I saw, that the size of pre miRNA and the stemloop primer is 103 bp - however considering the 6 bp added to the fw primer and that the rev primer will not amplify the last 10 bp of the stemloop primer the maximum amplicon size should be 99 bp and not 120-150 bp (I hope I got this right) .

The size of your amplicon you want to amplify (including the 6 5'prime bases of the fw primer which do not belong to the sequence of the miRNA) should be 84 bp which fits more or less to the peak of your melting curve.

I think your primers looking fine (I don't see that your fw primer is extending in any upstream region, especially as this primer is at the 5' prime end of the miRNA (mature and precursor miRNA) and it matches perfectly with you mature miRNA. I would consinder any mispriming in the precursor miRNA as unlikely. Your reverse primer is binding only in the stemloops sequence - I would consider mispriming as unlikely.

But what I have seen is that your primers might misprime (if you are unlucky) in another mRNA - it is the DNAAF1 gene creating an amplicon of 119 bp fitting to your observation (there are other hits but these amplicons are so big that I don't thing they would show up during qPCR). For this reason I was asking which kit do you use. I will attach a pictue of the primer blast search. If this is really the problem you might want to change the method of miRNA isolation and/or you primer.

KR Dörthe

Hi Sima,

I spotted a mistake in my answer - the size of your amplicon you wanted to amplify should be 60 bp - not 84 as I previously answered. Sorry for the wrong calculation

@Dörthe Andrea Kesper

Hi

I extracted total Rna.

Thanks a million for your time

@Muhammad Waqas

Thanks for your time. I will check blast again