I tried to detect a short non-coding RNA (80nt) but taqman detection gave me very late ct (about ct 34-36) when I synthesize cDNA with specific primers. I wish to pre-amplify it before subject it to taqman or SYBR RT-PCR (hope to get earlier ct). Anyone has good pre-amplification protocols for miRNA or non-coding RNAs can share? thanks
Which cDNA syn. protocol did you follow, did you tailed the miRNA before reverse transcription? In some case of miRNA the 34-36 ct is not that bogus.
measuring miRNA might be impossible with RT-qPCR as their length is very small (
hi Ferenc, i normally follow primer specific protocol for low abundance genes, with NEB RTase and RNase Inhibitor and reverse specific primers. in this case i am looking at circular RNAs, so i didn't knw how tailing works. any suggestions?
thanks Asif, some problem is if the primers are detecting genes which are low abundance and late, the dilution curve can only make a few points right.
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