Hi Sneha.

If if it’s the kit I think it is I use it

The poly A tail and 5’ linker are ligated so that you can amplify cDNA made from ligated miRNA species using universal primers

This is particularly useful for miRNA because yields of purified RNA are invariably low and consequently qPCR is not that reliable or sensitive unless you amplify the cDNA derived from miRNA without the extra amplification step

Hi Sneha,

adding a Poly(a)-tail and an anchor region is performed so you can use the same generated cDNA to perform multiple assays (I assume). Traditional Taqman Assays required that you use the primer from the Assay-Kit to generate specific cDNA. So you always needed to perform the RT reaction for every individual assay and can't use it for anything else.

Best regards

Hi Sneha.

If if it’s the kit I think it is I use it

The poly A tail and 5’ linker are ligated so that you can amplify cDNA made from ligated miRNA species using universal primers

This is particularly useful for miRNA because yields of purified RNA are invariably low and consequently qPCR is not that reliable or sensitive unless you amplify the cDNA derived from miRNA without the extra amplification step

Thank you very much for providing the explanation. I think I now better understand the underlying significance of it now.

I would like to extend your question. The system uses the polyadenylated miRNA as a template for RT once the universal primer binds to the poly-A tail. However, when you use total RNA, including miRNA, you would also have mRNA, which is polyadenylated as well. Does the primer have another characteristic specific for miRNA or the mRNA is used as a template too?

Hi Ana,

you are not only adding a poly(A) tail to the miRNA but also an adaptor region which binds a specific primer that is included in the kit, much like in a RACE-experiment. Therefore you won't amplify mRNA which of course is also present in your reaction.

https://tools.thermofisher.com/content/sfs/manuals/100027897_TaqManAdv_miRNA_Assays_UG.pdf

Best regards

Thanks Soner and Ana. Good qualifications to my basic answer