Limited effect of PCR ramp rate on qPCR Ct values: an exploratory study.

BACKGROUND

Quantitative real-time PCR (qPCR) is highly sensitive to technical parameters, including thermal cycler ramp rate (RR). We systematically evaluated the effect of slow, intermediate and fast RR on cycle threshold (Ct) values using a commercial TaqMan™ SNP assay and two housekeeping genes, GAPDH and PPIA.

METHODS AND

RESULTS

Eleven heterozygous DNA samples and three RNA samples were analyzed across two independent runs per RR. Across genotyping and gene expression assays, no consistent effect of qPCR RR on Ct values was observed. Intra-run analyses identified isolated statistically significant or borderline significant differences for the genotyping FAM-labeled probe and PPIA and GAPDH expression assays; however, these findings were not consistently reproduced across runs or pairwise comparisons. Overall, the observed effects appeared assay- and run-dependent rather than attributable to a consistent influence of RR.

CONCLUSIONS

These findings suggest that PCR RR does not have a consistent effect on Ct values across the evaluated genotyping and gene expression assays. The isolated differences observed between RRs were not consistently reproduced across runs. Larger studies involving more assays and samples are needed to determine whether PCR RR has a meaningful impact on qPCR performance and reproducibility under different experimental conditions.