What would make you reject a qPCR run even when the sample Cq values look plausible?

Why

A run is judged on its controls and curve quality first, and on sample values last. MIQE 2.0 lists positive, negative, no-template and no-RT controls as indispensable. A no-template control with probe signal in a diagnostic assay means the run should not be reported.

In research, a dye-based no-template signal may be a primer dimer, which a melt curve can show, but it still has to be explained. A positive control that shifts by more than its historical spread suggests a reagent, pipetting or instrument problem that affects every well. If the run carries a standard curve, an efficiency outside the lab's acceptance range or poor linearity undermines the quantities.

The curves themselves matter. Flattened or late-rising curves in some samples but not the positive control suggest inhibition. Inconsistent baselines suggest the threshold was set badly.

For example, sample Cq values of 23 to 27 can look normal while the no-RT controls sit at Cq 28. That means much of the signal could be genomic DNA. The point is that a wrong answer often looks reasonable.

Acceptance criteria should be written before the run, so the decision is not made after seeing the result.