Adrián Ruiz-Vilalba1, Bep van Pelt-Verkuil2, Quinn Gunst1, Maurice van den Hoff1, Jan Ruijter1
1Department of Anatomy, Embryology and Physiology, Academic Medical Centre (AMC), Amsterdam, The Netherlands;
2Department of Innovative Molecular Diagnostics, University of Applied Sciences, Leiden, the Netherlands
A recent comparison of qPCR data analysis methods showed that some of the amplification curve analysis methods perform better than the classic standard curve and Cq approach on indicators like variability and sensitivity. In this comparison the possibility to characterize the amplification curve and thus assess its quality remained under-exposed. To this end, different datasets have been compared. Our results show that for a significant fraction of the genes, low initial target concentrations lead to the amplification of artifacts independently of the primer specificity. These non-specific amplification curves are indistinguishable from those resulting in the correct products; they show similar baseline, PCR efficiency and plateau fluorescence behaviours. The validation of specific amplification curves requires a quality control in which the design of the plate, a melting curve analysis (MCA) and electrophoresis gels are combined. In addition, our data suggest that the relative concentration of the template in the cDNA input and of the primers determines the appearance of the PCR artifacts. Unexpectedly, the presence of non-template foreign cDNA seems to be an essential requirement for the amplification of the correct specific qPCR target.
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