qPCR: multiple melt peaks

1. Ranked causes

   1. Non-specific amplification from primers (anneal too low / design)Extra peak shrinks when anneal is raised 2–3 °C.
   2. Primer-dimer peak at low Tm alongside specific productLow-Tm peak present in NTC as well as samples.
   3. Contaminating template producing a second ampliconSecond peak matches a known off-target size on gel; fresh reagents help.
   4. Heterozygous indel / isoform mix in the sample biologyTwo specific-Tm peaks stable across clean NTCs and replicate preps.

2. Recovery steps

   1. Raise annealing temperature 2–3 °C and re-check meltOften removes non-specific peaks without losing the true product.
   2. Run NTC melt; if low-Tm peak matches samples, redesign or lower primer concentrationPrimer-dimer is diagnosed when NTC shares the extra peak.
   3. Spot-check on a gel or switch to a hydrolysis-probe chemistryConfirms whether extra fluorescence is a real second amplicon.

3. Controls first

   • Does the NTC also show one of the extra melt peaks?If yes: Primer-dimer or contamination — not sample isoform biology.

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