i have isolated total RNA from PBMCs and then synthesized cDNA from this total RNA. Now i want to check the miRNA expression in these cDNA with TaQman probes. please suggest. Is there is any probability that it will it miRNA form total cDNA in Real time PCR?
I am not sure by what you mean "total cDNA", there's no sub-species of cDNA as there are for RNA (miRNA, mRNA, ribosomal RNA, etc).
Did you use a miRNA column-based approach to purify your RNA? The traditional RNA columns will not co-purify miRNA fractions.
Assuming you did, you will need a cDNA sythesis method compatible with your probe-based approach.
https://www.thermofisher.com/order/catalog/product/4366596
After you made your cDNA that is compatible with your probe chemistry, you can use your Taqman probes.
Honestly, i think the taqman probes are overrated as the traditional SYBR green-based methods can also detect many miRNAs that are abundant. One can design dozen of primers that have similar sensitivity and specificity at a small fraction of a probe cost. However, probe-based methods have increased specificity for highly homologous family of miRNAs (let7s) which makes them superior in certain scenarios.
Hello Vikas
I study the miRNA epression from peripheral blood cells. If the total RNA from PBMC was perfomed with a kit only for RNA and not included the miRNA in its protocol, may be the microRNAs were discarded. The microRNA is a little hard to precipitate in alcohol, because it is a segment very short. For this reason some commercial kit (mirVana) have a special column and different steps using alcohol to recovery RNA and/or microRNAs.
The PCR from your cDNA will show a unreliable miRNA amplification.
Best regards
Hello Vikas,
Hope the attachment will be helpful for you. for my experiments i usually prefer SYBR green rather than other options.
Best regards
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