Maurice J.B. van den Hoff1, Quinn D. Gunst1, Adrian Ruiz-Villalba2, Carl Wittwer3, Jan M. Ruijter1
1) Amsterdam UMC, location AMC, Depart. Medical Biology, Amsterdam, The Netherlands;
2) Foundation of Applied Medical Research, University of Navarra, Pamplona, Spain;
3) University of Utah Health Sciences Center, Department of Pathology, Salt Lake City, UT, USA;
Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analysis of the results of 101 validated SybrGreen I-based assays for genes related to the Wnt-pathway in 5 different cardiac compartments frequently showed the amplification of nonspecific products, most probably primer-dimers. A detailed survey of these data revealed that the occurrence of nonspecific products is not related to Cq value or the PCR efficiency. qPCRs amplifying both specific and non-specific products can easily be identified when a melting curve analysis is performed. Currently, qPCRs that amplify both the specific and (a) nonspecific product(s) need to be excluded from further analysis because the quantification result is meaningless.
A model was developed, allowing the quantification of a qPCR in which the correct product together with additional off-target products is amplified. This model is based on the analysis of the melting peaks and the assignment of the total fluorescence at the end of the reaction to either the correct product or to other products. The fraction of fluorescence due to the amplification of the correct product can then be used to correct the quantification result (Cq value or target quantity, N0) that was derived from the observed amplification curve.
This correction method, and a program to analyze melting curves, was tested for the 101 different validated qPCR assays in different biological tissues and for model experiments with known concentrations of different products. The results of these tests show improvement of the sensitivity of SybrGreen I-based assays and avoid erroneous conclusion.
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