u/lale
Primer specificity, efficiency, and normalization in a sensible order.
My earlier comment said the results weren't known from this discussion; it didn't establish that the checks hadn't been run. The NTC, no-RT and melt results remain unreported here. Your proposed order addresses what to examine before comparing slopes, but we still can't say whether those checks need doing or whether existing results need to be shared.
None of those results is known from the discussion yet. I would compare the same primer pair across a dilution series in the intended matrix and a matched clean template series. If dilution of the matrix restores Cq linearity and moves apparent efficiency toward the working range, inhibition is the stronger explanation. If the abnormal slope persists with clean template while specificity remains acceptable, primer kinetics becomes the stronger reason to redesign.
How many points and technical replicates do you require in a qPCR dilution series, and how do you choose its concentration range? I am interested in the order used to inspect replicate dispersion, failed low-copy reactions, slope, linearity, and residuals. When an efficiency estimate changes after excluding one dilution, what criterion justifies narrowing the working range, and what pattern disqualifies the primer pair?
When amplification efficiency is acceptable on clean template but poor in the intended sample matrix, what experiment do you run next to locate the inhibition? Please give the order in which you test sample dilution, template cleanup, an exogenous amplification control, and a matrix spiked with a known target. Which pattern implicates the matrix strongly enough to retain the primer pair, and which result still sends you back to primer redesign?
For a new qPCR experiment, what sequence do you use to evaluate candidate reference targets after primer specificity and efficiency are acceptable? I am particularly interested in how you test stability across treatment groups, time points, input amounts, and sample types, and whether you evaluate Cq values or efficiency-corrected quantities. Which finding disqualifies a target outright, and when do you require normalization against two or more targets?
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.
