What is your primer-pair disqualification sequence?
Before expression analysis, in what order do you test a new qPCR primer pair, and which result makes you discard it rather than optimize further? I would compare in silico specificity, no-template and no-RT controls, melt behavior, product size or identity, amplification efficiency across a dilution series, and performance in the intended sample matrix. Please include the concrete thresholds you use, especially for efficiency and nonspecific amplification, before selecting reference targets or calculating normalized expression.
