What range of Ct values should I expect to see when using TaqMan qPCR with microRNA? What is the maximum acceptable Ct value for this kind of PCR, e.g. 30?
Hi,
The range of Ct values will depend on the quality of your sample and the level of expression of the microRNAs in question.
I haven't used the Advanced microRNA chemistry yet but I would expect you should see similar Ct's to the 'old' miR assays. I've seen anywhere from 17-18 cycles for high expression miRs to 35+ cycles for low expressors.
It's worth mentioning that all qPCR machines have limits of detection, so check how many cycles you can reliably go to on the particular machine you are using before disregarding data. 36-38 cycles is a usual upper limit though.
Hi Alisha
Totally in agreement with Benjamin. In my particular experience, and because we are dealing with miRNAs in biofluids, we extend the limit up to 40. For those low-expressed miRNAs I would also advice you to perform 45 amplification cycles in your qPCR machine.
Also in our experience, Taqman and other detection methods such as Sybrgreen performed similarly in miRNA detection.
Best
If possible, you want Ct values above about 16 and below 35 so you aren't at the detection limit for the machine.
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