A Refined, Rapid and Reproducible High Resolution Melt (HRM)-based Method Suitable for Quantification of Global LINE-1 Repetitive Element Methylation

Overview

Journal BMC Res Notes

Publisher Biomed Central

Specialties Biology
General Medicine

Date 2011 Dec 30

PMID 22204640

Citations 23

Authors

M Yat Tse

Janet E Ashbury

Nora Zwingerman

Will D King

Sherry Am Taylor

Stephen C Pang

Affiliations

Soon will be listed here.

Abstract

Background: 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.

Findings: 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.

Conclusions: 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.

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