靶向甲基化分析：从亚硫酸氢盐处理到定量。

Targeted Methylation Analyses: From Bisulfite Treatment to Quantification.

机构信息

Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

Department of Pathology, Portuguese Oncology Institute of Porto (IPOP), Porto, Portugal.

出版信息

Methods Mol Biol. 2021;2195:167-180. doi: 10.1007/978-1-0716-0860-9_12.

DOI:10.1007/978-1-0716-0860-9_12

PMID:32852764

Abstract

DNA methylation constitutes the most studied epigenetic mechanism, regulating gene expression in several physiological and pathological states. Targeted methylation polymerase chain reaction (PCR)-based analyses are among the most universal and commonly used techniques in research. They can be of use for validating methylation-based biomarkers to include in clinical practice. Optimal execution and interpretation of data is fundamental for achieving accurate and reproducible results.In this chapter we describe the backbone procedures behind targeted methylation analyses: bisulfite conversion and downstream PCR-based techniques, including real-time quantitative methylation-specific PCR (qMSP) and high-resolution melting (HRM) methylation-sensitive analyses. Specifically, we give details about the protocol, discuss the pros and cons of these methodologies, and give practical tips for achieving optimal results and for troubleshooting.

摘要

DNA 甲基化是研究最多的表观遗传机制之一，它在多种生理和病理状态下调节基因表达。基于靶向甲基化聚合酶链反应（PCR）的分析是研究中最普遍和常用的技术之一。它们可用于验证纳入临床实践的基于甲基化的生物标志物。为了获得准确和可重复的结果，最佳执行和解释数据是至关重要的。在本章中，我们描述了靶向甲基化分析的基本程序：亚硫酸氢盐转化和下游基于 PCR 的技术，包括实时定量甲基化特异性 PCR（qMSP）和高分辨率熔解（HRM）甲基化敏感分析。具体来说，我们详细介绍了该方案，讨论了这些方法的优缺点，并提供了实现最佳结果和解决问题的实用技巧。