Tse M Yat, Ashbury Janet E, Zwingerman Nora, King Will D, Taylor Sherry Am, Pang Stephen C
Department of Anatomy and Cell Biology, Queen's University, Kingston, ON, Canada.
BMC Res Notes. 2011 Dec 28;4:565. doi: 10.1186/1756-0500-4-565.
The methylation of DNA is recognized as a key mechanism in the regulation of genomic stability and evidence for its role in the development of cancer is accumulating. LINE-1 methylation status represents a surrogate measure of genome-wide methylation.
Using high resolution melt (HRM) curve analysis technology, we have established an in-tube assay that is linear (r > 0.9986) with a high amplification efficiency (90-105%), capable of discriminating between partcipant samples with small differences in methylation, and suitable for quantifying a wide range of LINE-1 methylation levels (0-100%)--including the biologically relevant range of 50-90% expected in human DNA. We have optimized this procedure to perform using 2 μg of starting DNA and 2 ng of bisulfite-converted DNA for each PCR reaction. Intra- and inter-assay coefficients of variation were 1.44% and 0.49%, respectively, supporting the high reproducibility and precision of this approach.
In summary, this is a completely linear, quantitative HRM PCR method developed for the measurement of LINE-1 methylation. This cost-efficient, refined and reproducible assay can be performed using minimal amounts of starting DNA. These features make our assay suitable for high throughput analysis of multiple samples from large population-based studies.
DNA甲基化被认为是调节基因组稳定性的关键机制，其在癌症发生中作用的证据正在不断积累。LINE-1甲基化状态代表全基因组甲基化的替代指标。
利用高分辨率熔解（HRM）曲线分析技术，我们建立了一种管内检测方法，该方法呈线性（r>0.9986），扩增效率高（90-105%），能够区分甲基化存在微小差异的参与者样本，适用于定量广泛范围的LINE-1甲基化水平（0-100%），包括人类DNA中预期的生物学相关范围50-90%。我们已优化该程序，每个PCR反应使用2μg起始DNA和2ng亚硫酸氢盐转化的DNA进行检测。批内和批间变异系数分别为1.44%和0.49%，支持该方法具有高重现性和精密度。
总之，这是一种为测量LINE-1甲基化而开发的完全线性、定量的HRM PCR方法。这种经济高效、精细且可重复的检测方法可使用极少量的起始DNA进行。这些特性使我们的检测方法适用于对来自大型人群研究的多个样本进行高通量分析。
