Hi,

Both pri-miRNA and mature miRNA can be quantified using either SYBR green-based approach or Taqman assay, the latter of which is generally more specific and expensive.

For SYBR green quantitation of mature miRNA, you first treat the total RNA or small RNA-enriched fraction with poly(A) polymerase, reverse transcribe using a universal primer (which hybridizes the elongated A tail) and PCR amplify with a pair of miRNA-specific forward primer and universal reverse primer.

For Taqman quantitation of mature miRNA, you need specific Taqman probe, stem-loop RT primer and PCR primers for each miRNA, which are nowadays pre-designed and sold in ready-to-use formulations by Thermo Fischer  Scientific.

SYBR green quantitation of pri-miRNA is not much different from mRNA measurement, except for the steady-state levels of pri-miRNAs are generally low compared to mRNAs and therefore their Ct value often exceeds 30, which necessitate rigorous control experiments and validation (e.g. Does the signal increase upon knockdown/knockout of miRNA biogenesis factor such as Drosha/DGCR8 in animals and DCL1 in plants?).

Hi Malay,

Well, my suggestion is not that cheap but at least is sensitive and fairly robust! I would use TaqMan™ Pri-miRNA Assays! It takes a bit time after you order (they are "Made To Order").

Cheers

Ali

Thanks Dr Gheinani,

I think using the mature miRNA primers by syber-green detects both pri-miRNAs and mature miRNAs. So I was hoping to design a primer for pri-miRNA excluding the sequence of mature miRNA and use it for syber green qRT-PCR. Thus subtracting the relative levels of the former with later from two separate qRT-PCRs with same internal control (U6) may give us the relative expression of mature miRNA only.

Please suggest...!!