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Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM)

Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes...

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Autores principales: Samsø Mathiasen, Sally, Bińkowski, Jan, Kjeldsen, Tina, Wojdacz, Tomasz K., Hansen, Lise Lotte
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9447921/
https://www.ncbi.nlm.nih.gov/pubmed/36067175
http://dx.doi.org/10.1371/journal.pone.0273058
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author Samsø Mathiasen, Sally
Bińkowski, Jan
Kjeldsen, Tina
Wojdacz, Tomasz K.
Hansen, Lise Lotte
author_facet Samsø Mathiasen, Sally
Bińkowski, Jan
Kjeldsen, Tina
Wojdacz, Tomasz K.
Hansen, Lise Lotte
author_sort Samsø Mathiasen, Sally
collection PubMed
description Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the amplification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is hampered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of samples using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays’ specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves.
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spelling pubmed-94479212022-09-07 Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM) Samsø Mathiasen, Sally Bińkowski, Jan Kjeldsen, Tina Wojdacz, Tomasz K. Hansen, Lise Lotte PLoS One Lab Protocol Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the amplification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is hampered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of samples using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays’ specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves. Public Library of Science 2022-09-06 /pmc/articles/PMC9447921/ /pubmed/36067175 http://dx.doi.org/10.1371/journal.pone.0273058 Text en © 2022 Samsø Mathiasen et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Lab Protocol
Samsø Mathiasen, Sally
Bińkowski, Jan
Kjeldsen, Tina
Wojdacz, Tomasz K.
Hansen, Lise Lotte
Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM)
title Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM)
title_full Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM)
title_fullStr Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM)
title_full_unstemmed Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM)
title_short Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM)
title_sort methylation levels assessment with methylation-sensitive high-resolution melting (ms-hrm)
topic Lab Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9447921/
https://www.ncbi.nlm.nih.gov/pubmed/36067175
http://dx.doi.org/10.1371/journal.pone.0273058
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