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Removal of artifact bias from qPCR results using DNA melting curve analysis. / Ruijter, Jan M.; Ruiz-Villalba, Adrian; van den Hoff, Axel J. J. et al.

In: FASEB journal, Vol. 33, No. 12, 2019, p. 14542-14555.

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Harvard

Ruijter, JM, Ruiz-Villalba, A, van den Hoff, AJJ, Gunst, QD, Wittwer, CT & van den Hoff, MJB 2019, 'Removal of artifact bias from qPCR results using DNA melting curve analysis', FASEB journal, vol. 33, no. 12, pp. 14542-14555. https://doi.org/10.1096/fj.201901604R

APA

Vancouver

Ruijter JM, Ruiz-Villalba A, van den Hoff AJJ, Gunst QD, Wittwer CT, van den Hoff MJB. Removal of artifact bias from qPCR results using DNA melting curve analysis. FASEB journal. 2019;33(12):14542-14555. doi: 10.1096/fj.201901604R

Author

Ruijter, Jan M. ; Ruiz-Villalba, Adrian ; van den Hoff, Axel J. J. et al. / Removal of artifact bias from qPCR results using DNA melting curve analysis. In: FASEB journal. 2019 ; Vol. 33, No. 12. pp. 14542-14555.

BibTeX

@article{b7ba401b4cae407b9b6b13990ea38c48,
title = "Removal of artifact bias from qPCR results using DNA melting curve analysis",
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.",
author = "Ruijter, {Jan M.} and Adrian Ruiz-Villalba and {van den Hoff}, {Axel J. J.} and Gunst, {Quinn D.} and Wittwer, {Carl T.} and {van den Hoff}, {Maurice J. B.}",
year = "2019",
doi = "10.1096/fj.201901604R",
language = "English",
volume = "33",
pages = "14542--14555",
journal = "FASEB journal",
issn = "0892-6638",
publisher = "FASEB",
number = "12",

}

RIS

TY - JOUR

T1 - Removal of artifact bias from qPCR results using DNA melting curve analysis

AU - Ruijter, Jan M.

AU - Ruiz-Villalba, Adrian

AU - van den Hoff, Axel J. J.

AU - Gunst, Quinn D.

AU - Wittwer, Carl T.

AU - van den Hoff, Maurice J. B.

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85075956760&origin=inward

UR - https://www.ncbi.nlm.nih.gov/pubmed/31682470

U2 - 10.1096/fj.201901604R

DO - 10.1096/fj.201901604R

M3 - Article

C2 - 31682470

VL - 33

SP - 14542

EP - 14555

JO - FASEB journal

JF - FASEB journal

SN - 0892-6638

IS - 12

ER -

ID: 7771564