Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Independent Clinical Epigenetics Laboratory, Pomeranian Medical University in Szczecin, Szczecin, Poland.
PLoS One. 2022 Sep 6;17(9):e0273058. doi: 10.1371/journal.pone.0273058. eCollection 2022.
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.
检测与疾病相关的 DNA 甲基化变化可为个性化患者护理提供具有临床相关性的信息。甲基化敏感高分辨率熔解（MS-HRM）是一种用于测量甲基化变化的方法，已在诊断环境中使用。该方法使用一组引物启动甲基化和非甲基化模板的扩增。因此，MS-HRM 中使用 PCR 偏倚现象会阻碍对甲基化水平的定量。已经提出了一些方法来使用高分辨率熔解（HRM）曲线计算样品的甲基化水平。然而，MS-HRM 中使用的甲基化计算的局限性尚未得到系统和全面的评估。我们使用 HRM 曲线的 AUC（曲线下面积）和最小二乘逼近（LSA）建立了一个程序，该程序允许我们在 MS-HRM 实验中推断甲基化水平，并评估该程序在特定甲基化水平测量方面的局限性。所开发的程序允许在一定程度上使用 HRM 曲线估计甲基化水平。
