Format

Send to

Choose Destination
FASEB J. 2019 Dec;33(12):14542-14555. doi: 10.1096/fj.201901604R. Epub 2019 Nov 2.

Removal of artifact bias from qPCR results using DNA melting curve analysis.

Author information

1
Department of Medical Biology, Amsterdam University Medical Centers, Academic Medical Center (AMC), Amsterdam, The Netherlands.
2
Centre for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain; and.
3
Department of Pathology, University of Utah Medical School, Salt Lake City, Utah, USA.

Abstract

Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analyses using validated Sybr Green I-based assays regularly amplify both the correct product and an artifact. Amplification of more than 1 product can be recognized when melting curve analysis is performed after the qPCR. Currently, such reactions need to be excluded from further analysis because the quantification result is considered meaningless. However, when the fraction of the fluorescence associated with the correct product can be determined, the quantitative result of the qPCR analysis can be corrected. The main assumptions of this correction model are: 1) the melting peak of the correct product can be identified, 2) the PCR efficiencies of all amplified products are similar, 3) the relative size of the melting peaks reflects the relative concentrations of the products, and 4) the relative concentrations do not change as the reaction reaches plateau. These assumptions were validated in a series of model experiments. The results show that the quantitative results can be corrected. Implementation of a correction for the presence of artifact amplification in the analysis of qPCR data leads to more reliable quantitative results in qPCR experiments.-Ruijter, J. M., Ruiz-Villalba, A., van den Hoff, A. J. J., Gunst, Q. D., Wittwer, C. T., van den Hoff, M. J. B. Removal of artifact bias from qPCR results using DNA melting curve analysis.

KEYWORDS:

PCR efficiency; RT-qPCR; amplification curve analysis; artifact amplification; qPCR data analysis

PMID:
31682470
DOI:
10.1096/fj.201901604R

Supplemental Content

Full text links

Icon for Atypon
Loading ...
Support Center