PCR Efficiency Greater Than 120% with Inconsistent ΔCq (PCR (Sigma Guide))

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Accepted Answer

PCR efficiency calculated from standard curve is greater than 120%; ΔCq between 10-fold dilutions is much less than expected 3.3 cycles (e.g., 1.5 cycles); standard curve gradient indicates abnormally high efficiency Common causes: 1) Excessive primer-dimer formation particularly in low concentration samples 2) Pipetting is inaccurate causing errors in serial dilutions 3) Inhibition present in samples 4) Low concentration samples contain additional signal from primer dimers Solutions: 1) Evaluate dissociation/melt curve plot to identify primer dimers (look for lower Tm peak in low concentration samples) 2) Try lower primer concentrations to reduce primer-dimer 3) Test pipette calibration and ensure accurate pipetting technique 4) Check ΔCq between dilutions - if decreasing, indicates inhibitor (confirm with SPUD assay) 5) Purify sample or use higher dilution in the assay 6) With RT-PCR: use less RT enzyme, adopt 2-step protocol, or use higher RT incubation temperature