Premium
Removal of artifact bias from qPCR results using DNA melting curve analysis
Author(s) -
Ruijter Jan M.,
RuizVillalba Adrian,
Hoff Axel J. J.,
Gunst Quinn D.,
Wittwer Carl T.,
Hoff Maurice J. B.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.201901604r
Subject(s) - melting curve analysis , artifact (error) , sybr green i , real time polymerase chain reaction , quantitative analysis (chemistry) , product (mathematics) , analytical chemistry (journal) , melting temperature , dna , chemistry , chromatography , biology , materials science , mathematics , biochemistry , geometry , neuroscience , gene , composite material
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. FASEB J. 33, 14542‐14555 (2019). www.fasebj.org