qPCR: poor efficiency

1. Ranked causes

   1. Inhibitors in template or standards (carry-over from prep)Efficiency improves after dilution or clean-up of the same samples.
   2. Standard dilution series inaccurate (pipetting / diluent)R² poor or points zigzag; remade dilutions straighten the curve.
   3. Primer concentration or amplicon design suboptimalSame standards give good efficiency with a different validated assay.
   4. Baseline/threshold settings distort early or late cyclesManual threshold in exponential phase restores efficiency near 100%.

2. Recovery steps

   1. Remake the standard curve with fresh serial dilutions (new tips each step)Dilution error is the most common efficiency failure.
   2. Dilute samples/standards further to outrun inhibitors; compare efficiencyInhibition presents as efficiency << 100% that recovers on dilution.
   3. Re-analyse with threshold set in the exponential phase for all wellsAnalysis artefacts can mimic chemistry problems.

3. Controls first

   • Is the standard-curve R² also poor, or only the efficiency slope off?If yes: Dilution series or pipetting — remake standards before redesigning primers.

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