Sigalotti Luca, Covre Alessia, Colizzi Francesca, Fratta Elisabetta
Istituto di Patologia Clinica, Azienda Sanitaria Universitaria Integrata, Udine, Italy.
Center for Immuno-Oncology, Division of Medical Oncology and Immunotherapy, Department of Oncology, University Hospital of Siena, Siena, Italy.
Methods Mol Biol. 2019;1909:137-162. doi: 10.1007/978-1-4939-8973-7_11.
Aberrant DNA methylation of cell-free circulating DNA (cfDNA) has recently gained attention for its use as biomarker in cancer diagnosis, prognosis, and prediction of therapeutic response. Quantification of cfDNA methylation levels requires methods with high sensitivity and specificity due to low amounts of cfDNA available in plasma, high degradation of cfDNA, and/or contamination with genomic DNA. To date, several approaches for measuring cfDNA methylation have been established, including quantitative methylation-specific PCR (qMSP), which represents a simple, fast, and cost-effective technique that can be easily implemented into clinical practice. In this chapter, we provide a detailed protocol for SYBR Green qMSP analysis which is currently used in our laboratory for cfDNA methylation detection. Useful information regarding successful qMSP primers design are also provided.
游离循环DNA（cfDNA）的异常DNA甲基化最近因其作为癌症诊断、预后和治疗反应预测的生物标志物而受到关注。由于血浆中可获得的cfDNA量低、cfDNA高度降解和/或受基因组DNA污染，cfDNA甲基化水平的定量需要具有高灵敏度和特异性的方法。迄今为止，已经建立了几种测量cfDNA甲基化的方法，包括定量甲基化特异性PCR（qMSP），这是一种简单、快速且经济高效的技术，可轻松应用于临床实践。在本章中，我们提供了SYBR Green qMSP分析的详细方案，该方案目前在我们实验室用于cfDNA甲基化检测。还提供了有关成功设计qMSP引物的有用信息。
