Lale

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.