Department of Translational Research and New Technologies in Medicine and Surgery, Division of Medical Genetics, University of Pisa, Pisa, Italy.
PLoS One. 2013;8(1):e52501. doi: 10.1371/journal.pone.0052501. Epub 2013 Jan 11.
There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing.
人们越来越关注开发经济有效的技术来定量检测 DNA 甲基化生物标志物。我们使用甲基化敏感高分辨率熔解（MS-HRM）和焦磷酸测序法分析了 90 例手术切除的结直肠癌细胞组织中 APC 和 CDKN2A 启动子的甲基化情况。与焦磷酸测序相比，MS-HRM 是一种成本较低的技术，但通常更受限制，因为它给出的是甲基化估计范围，而不是单一值。在这里，我们开发了一种使用 MS-HRM 获得单个估计值（而不是范围）的方法，并将以这种方式获得的值与使用焦磷酸测序的金标准定量方法获得的值进行了比较。我们使用已知甲基化/未甲基化比例（0％、12.5％、25％、50％、75％和 100％甲基化）的标准品来构建一条内插曲线，以获得每个样本的最佳甲基化程度估计值。我们观察到两种技术的甲基化谱相似，且相关性系数较高。总的来说，我们的新方法允许将 MS-HRM 用作定量测定，其结果可与焦磷酸测序获得的结果相媲美。
