I am using Taqman microRNA assay to quantify miRNAs. Do you have to quantify the cDNA before going for qRT-PCR? The initial concentration of total RNA is 10ng/15uL reaction. What would be to possible concentration of cDNA after the reverse transcription reaction?
Quantifying cDNA is difficult unless using a fluorometric method specific for single-stranded DNA.
Your cDNA is thus perhaps 0.667 ng/uL (if you are saying your RT reactions were each 10 ng/15 uL).
Assuming no inhibitory characteristics of your cDNA on the qPCR, your samples are near ideal concentrations already for use directly in qPCR. They undergo additional dilution already by being placed into each qPCR.
Most cDNA starts out at 20 to 50 ng/uL, you are starting at 0.667 ng/uL - so you are on the dilute side already. But, if your cDNA is good, and non-inhibitory, you should be fine as is. Each uL of your 0.667 ng/uL sample represents ~33 eucaryotic transcriptomes worth of total RNA.
HI anup VINAY here man...
generally in our lab we dilute the cdna by 200 times dilution for estimating the mirna......
Dear Anup,
According the technical advisory from Life Technologies (currently Thermo Fischer Scientific) the amount of RNA in cDNA for qPCR of miRNA it is between 10 and 20 ng/uL. I personally used the concentration of 10 ng/uL and did not have any problem with it. Thus, according to them the sensibility of the assay range from 1 to 20 ng/uL of RNA. I obtained these information when I entered in contact with them since some years ago.
I hope that it helps you.
Pedro
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