qPCR: late / no ct

1. Ranked causes

   1. Template degraded, too dilute, or RT failure (RNA input)Reference gene also late/absent; RIN/cDNA QC poor after the same prep.
   2. Master mix / polymerase lot inactive (storage, freeze-thaw)All assays on the plate fail including the kit positive control.
   3. Primer/probe design or concentration wrong for this assayPositive-control gDNA/cDNA fails while a different validated assay works on the same plate.
   4. Annealing temperature or cycling profile offGradient PCR or −3 °C anneal restores amplification for this primer pair.
   5. Thermocycler block calibration driftSame mix succeeds on a second instrument.

2. Recovery steps

   1. Re-run with a verified positive-control template and fresh master-mix aliquotSplits template vs reagent vs instrument in one plate.
   2. Drop annealing temperature 3 °C and re-check amplification curvesCommon fix when primers are intact but binding is marginal.
   3. Quantify and dilute input into the validated dynamic rangeOver-dilute or inhibited templates present as late/no Ct.

3. Controls first

   • Did the positive-control / standard-curve wells also show late or no Ct?If yes: Master mix, primers, cycling, or instrument — not only this sample.
   • Did the NTC stay flat (no amplification)?If yes: Not contamination-driven signal; focus on template/assay chemistry.

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