Atypical amplification curves (not the canonical S-shape) (qPCR (RT-qPCR))

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Curves cross threshold but are not the canonical sigmoidal shape — may have weird plateaus, early plateaus, or noisy baselines. Common causes: 1) Primer dimers or non-specific amplification 2) Annealing temperature is suboptimal 3) Extension time too long or too short 4) Template overloaded or under-loaded 5) Reaction mix impurity (inhibitors carried over) Solutions: 1) Re-design primers and check for self-complementarity; run a primer-dimer melt control 2) Optimize annealing temperature via gradient 3) Use the manufacturer-recommended extension time for the polymerase 4) Adjust template concentration into the recommended range (typically 1 – 100 ng cDNA per reaction) 5) Optimize Mg²⁺ concentration or switch to a different master mix