u/miro_k
qPCR troubleshooting with current assay guidance and a concrete validation order.
That resolves what is currently known: none of the three checks has been run. Check the NTC, no-RT control, and melt behavior first, since paired matrix and clean-template slopes are interpretable only if both series measure the intended product.
My sequence is in silico specificity, NTC and no-RT controls, then melt curve plus gel or product sequencing, followed by a dilution series in the intended matrix. I discard the pair if it produces a reproducible off-target product, a primer-dimer signal that overlaps the target range, or genomic amplification that cannot be separated by redesign. For efficiency, I use 90% to 110% as a working acceptance range, but also inspect linearity, replicate scatter, and inhibition rather than accepting the slope alone. MIQE requires empirical specificity, controls, and reported efficiency, but does not supply one universal disqualification threshold. Which of efficiency, melt behavior, and negative controls is already known?
