Is there a universal primer kit which is comercially available for all known miRNA for RT-PCR?
Universal primers for miRNA RT-PCR require the introduction of fixed sequences. A 3' primer can be added by polyadenylating the RNA using PolyA Polymerase and then reverse-transcribing using an oligo(dT) with a fixed sequence end. This can then be used to anneal a universal specific 3' primer in the PCR step. The 5' PCR primer in such a system is usually derived from the miRNA sequence itself. This way you can get a miRNA-specific readout. I am not aware of an easy way to thus amplify ALL miRNAs. An example of such a kit would be QIAGEN miScript
An alternative is ligating 3' and 5' linkers (usually DNA-RNA chimeras) using T4 RNA Ligase. This is employed in making miRNA libraries for next generation sequencing - check the various manufacturers' websites (e.g. Illumina) for recommendation.
If you can be more specific about your experimental design and question you are trying to address, I can perhaps give more specific suggestions.
Good luck!
Anna
Thanks Anna very much. In fact I have the mature sequence of many miRNA sequences and would like to confirm the results using RT-PCR. Since the miRNA sequences are conserved I imagine there are universal primers available for RT-PCR.
I have noticed some people using the miRNA precursor sequence to measure the expression level of the corresponding mature miRNA. Which is better to measure the expression level of the miRNA or the precursor of that miRNA?
Regards,
M. Yaish
As mature miRNAs are the active form of the miRNAs, investigating the mature miRNAs is of higher priority. Note that some precursor miRNAs are efficiently processed into their mature form, thus significantly decreasing the abundance of the precursor miRNA (and as a result compromising your conclusions). If you find a precursor miRNA with a low expression level, you would not be able to distinguish whether it is transcriptionally lowly expressed or efficiently processed into its mature form.
In addition to the Qiagen miScript kit, Invetrogen's Ncode Villo Sybr Green kit for miRNAs and the Exiqon miRNA qRT-PCR kit can be suitable for your experiments.
Hope this helps.
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