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Article

Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment

by
Loretta De Chiara
1,2,*,
Virginia Leiro-Fernandez
3,4,
Mar Rodríguez-Girondo
5,
Diana Valverde
1,2,
María Isabel Botana-Rial
3,4 and
Alberto Fernández-Villar
3,4
1
Department of Biochemistry, Genetics and Immunology, University of Vigo, 36310 Vigo, Spain
2
Centro de Investigaciones Biomédicas (CINBIO), Centro Singular de Investigación de Galicia, Universidad de Vigo, 36310 Vigo, Spain
3
Pulmonary Department, Hospital Álvaro Cunqueiro, EOXI Vigo, 36213 Vigo, Spain
4
PneumoVigo I +i Research Group, Sanitary Research Institute Galicia Sur (IIS Galicia Sur), 36213 Vigo, Spain
5
Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, 2300RC Leiden, The Netherlands
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(23), 9242; https://doi.org/10.3390/ijms21239242
Submission received: 29 September 2020 / Revised: 18 November 2020 / Accepted: 1 December 2020 / Published: 3 December 2020
(This article belongs to the Special Issue Epigenetic Mechanisms and Human Pathology)

Abstract

Different methodological approaches are available to assess DNA methylation biomarkers. In this study, we evaluated two sodium bisulfite conversion-dependent methods, namely pyrosequencing and methylation-specific qPCR (MS-qPCR), with the aim of measuring the closeness of agreement of methylation values between these two methods and its effect when setting a cut-off. Methylation of tumor suppressor gene p16/INK4A was evaluated in 80 lung cancer patients from which cytological lymph node samples were obtained. Cluster analyses were used to establish methylated and unmethylated groups for each method. Agreement and concordance between pyrosequencing and MS-qPCR was evaluated with Pearson’s correlation, Bland–Altman, Cohen’s kappa index and ROC curve analyses. Based on these analyses, cut-offs were derived for MS-qPCR. An acceptable correlation (Pearson’s R2 = 0.738) was found between pyrosequencing (PYRmean) and MS-qPCR (NMP; normalized methylation percentage), providing similar clinical results when categorizing data as binary using cluster analysis. Compared to pyrosequencing, MS-qPCR tended to underestimate methylation for values between 0 and 15%, while for methylation >30% overestimation was observed. The estimated cut-off for MS-qPCR data based on cluster analysis, kappa-index agreement and ROC curve analysis were much lower than that derived from pyrosequencing. In conclusion, our results indicate that independently of the approach used for estimating the cut-off, the methylation percentage obtained through MS-qPCR is lower than that calculated for pyrosequencing. These differences in data and therefore in the cut-off should be examined when using methylation biomarkers in the clinical practice.
Keywords: DNA methylation; bisulfite pyrosequencing; methylation-specific qPCR; cut-off; methylation biomarker; non-small cell lung cancer; cytological lymph node samples DNA methylation; bisulfite pyrosequencing; methylation-specific qPCR; cut-off; methylation biomarker; non-small cell lung cancer; cytological lymph node samples

Share and Cite

MDPI and ACS Style

De Chiara, L.; Leiro-Fernandez, V.; Rodríguez-Girondo, M.; Valverde, D.; Botana-Rial, M.I.; Fernández-Villar, A. Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment. Int. J. Mol. Sci. 2020, 21, 9242. https://doi.org/10.3390/ijms21239242

AMA Style

De Chiara L, Leiro-Fernandez V, Rodríguez-Girondo M, Valverde D, Botana-Rial MI, Fernández-Villar A. Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment. International Journal of Molecular Sciences. 2020; 21(23):9242. https://doi.org/10.3390/ijms21239242

Chicago/Turabian Style

De Chiara, Loretta, Virginia Leiro-Fernandez, Mar Rodríguez-Girondo, Diana Valverde, María Isabel Botana-Rial, and Alberto Fernández-Villar. 2020. "Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment" International Journal of Molecular Sciences 21, no. 23: 9242. https://doi.org/10.3390/ijms21239242

APA Style

De Chiara, L., Leiro-Fernandez, V., Rodríguez-Girondo, M., Valverde, D., Botana-Rial, M. I., & Fernández-Villar, A. (2020). Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment. International Journal of Molecular Sciences, 21(23), 9242. https://doi.org/10.3390/ijms21239242

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