Comparison study of MS-HRM and pyrosequencing techniques for quantification of APC and CDKN2A gene methylation.
Comparison study of MS-HRM and pyrosequencing techniques for quantification of APC and CDKN2A gene methylation.
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DOI：
10.1371/journal.pone.0052501
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Migliore L
中科院分区：
文献类型：
--
作者：
Migheli F;Stoccoro A;Coppedè F;Wan Omar WA;Failli A;Consolini R;Seccia M;Spisni R;Miccoli P;Mathers JC;Migliore L
人们对开发用于定量DNA甲基化生物标志物的成本效益技术越来越感兴趣。我们分析了90例手术切除的结直肠癌组织的APC和CDKN 2A启动子甲基化，使用甲基化敏感高分辨率熔解（MS-HRM）和焦磷酸测序。与焦磷酸测序相比，MS-HRM是一种更便宜的技术，但通常更受限制，因为它给出了一系列甲基化估计值，而不是单个值。在这里，我们开发了一种方法来获得单一的估计，而不是一个范围内，使用MS-HRM甲基化，并比较以这种方式获得的值与使用金标准定量方法焦磷酸测序。我们使用已知甲基化/未甲基化比率（甲基化的0%、12.5%、25%、50%、75%和100%）的标准品推导出插值曲线，以获得我们每个样品的甲基化程度的最佳估计值。我们观察到两种技术之间相似的甲基化谱和高相关系数。总的来说，我们的新方法允许MS-HRM被用作定量测定，其提供的结果与通过焦磷酸测序获得的结果相当。
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.
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