How do I improve telomere amplification PCR efficiency in monochrome multiplex qPCR?

28 July 2020 0 5K Report

I am following Cawthon 2009 method, with 2 targets amplified in one well. Single copy gene (albumin) amplification in the same well is great. As the machine used for this experiment is Roche LightCycler 480, raw fluorescence values are collected afterwards to be run through LinRegPCR application. It generates Cq values for each sample that is setup in triplicate. I already increased the number of cycles from 32 to 45, as shorter cycle number did not reach plateau phase at the end of the run. Still, telomere amplicon efficiency is low according to the LinRegPCR, although from the fluorescence values I don't see it that way. Fluorescence signal increases strongly to plateau at the end. Could it be the LinRegPCR settings?

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