一种用于临床样本中 CST6 启动子甲基化半定量测定的闭管甲基化敏感高分辨率熔解分析（MS-HRMA）。
A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.
机构信息
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, 15771, Greece.
出版信息
BMC Cancer. 2012 Oct 22;12:486. doi: 10.1186/1471-2407-12-486.
BACKGROUND
CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.
METHODS
We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.
RESULTS
The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.
CONCLUSIONS
The developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.
背景
CST6 启动子在癌症中高度甲基化，其检测可为乳腺癌患者提供重要的预后信息。本研究旨在开发一种用于检测 CST6 启动子甲基化的甲基化敏感高分辨率熔解分析（MS-HRMA）检测方法。
方法
我们设计了引物，这些引物在经亚硫酸氢钠（SB）处理后可扩增甲基化和非甲基化的 CST6 序列，并使用完全甲基化至非甲基化 SB 转化的基因组 DNA 的加标对照样品来优化检测方法。我们首先通过分析 36 个样本（试点培训组）评估了该检测方法，然后进一步分析了 80 个可手术乳腺癌患者的 FFPES（独立组）。对所有 116 个样本的 MS-HRMA 检测结果与甲基化特异性 PCR（MSP）进行了比较，结果具有可比性。
结果
该检测方法具有高度特异性和灵敏度，因为它可以检测到 1%甲基化 CST6 序列的存在，并且还可以对 CST6 启动子甲基化进行半定量估计。在 80 个 FFPE 中有 39 个（48.75%）的 CST6 启动子发生甲基化，样本之间的甲基化水平差异很大。当用两种检测方法分析所有样本时，MS-HRMA 和 MSP 的结果具有可比性。
结论
开发的 CST6 启动子甲基化 MS-HRMA 检测方法是一种闭管、高度敏感、具有成本效益、快速且易于实施的方法。与 MSP 相比，它在更短的时间内提供了可比的结果，同时还具有提供甲基化水平估计的优势。
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