HI Ahmed,

I completely agree with you on this. Although I do not have much experience on miRNA, I faced similar problem with viral RNA. Since viral mRNA are on on overdrive during infection, I was not able to get a PCR Ct value. Until one day my PI suggested that many times when target sequence is too abundant, the primers are not saturating in the PCR mix as they should be which results inconsistent Ct values. I think same is the case with miRNA wherein primers are almost the size of target. Hopefully it helps.

Best

Surya

Is is the same procedure like PCR. If DNA concentration is too high, PCR does not work. like they say 10ng to 40ng is ideal for PCRing and it also depends how much is your reaction mixture. Same way you need to adjust cDNA concentration if needed. Where it works, note down and keep the same dilution. I have not worked microRNA but the principle should be the same

Hi Ahmed

Totally agree with the opinions already showed. qPCR for miRNAs is very similar to the protocol applied to other RNAs. I experienced the same behavior like you described, where the cDNA must be diluted prior to qPCR.

For miRNAs from cells or tissues, I routinely use a dilution of 1/5 to 1/10 depending on the initial RNA concentration to ensure a final cDNA amount around 10-100 ng per reaction.

Note that if you are working with circulating miRNAs in biofluids like plasma or serum, you should also dilute the initial RNA sample prior RT reaction due to the presence of RT inhibitors.

Best of luck

Cheers. Paco

First you have to measure the RNA conc. then you decide...after that..the cDNA most of the time need to be diluted