Investigation of Reproducibility and Error Associated with qPCR Methods using Quantifiler® Duo DNA Quantification Kit*

Reproducibility of quantitative PCR results is dependent on the generation of consistent calibration curves via accurate volume transfers and instrument performance. A review of 14 standard curves, using two different QuantDuo® standard DNA lots, showed variability of cycle threshold values between assays were larger than those of the Internal PCR Control (IPC). This prompted a set of experiments designed to determine the source of variability. Results showed that error introduced during DNA addition to the plate resulted in little variation. A comparison of seven independent series demonstrated cycle threshold variation between dilutions was larger than the variation expected from repeated samples. Modeling the influence of pipette errors on dilution series accuracy indicated that a more rigorous approach to external calibration curve production is required and showed that improvement in calibration curve stability is expected if the pipette conditions are carefully chosen and ⁄ or a single validated curve is utilized as the calibrator.