A refined, rapid and reproducible high resolution melt (HRM)-based method suitable for quantification of global LINE-1 repetitive element methylation

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...

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Published inBMC research notes Vol. 4; no. 1; p. 565
Main Authors Tse, M Yat, Ashbury, Janet E, Zwingerman, Nora, King, Will D, Taylor, Sherry Am, Pang, Stephen C
Format Journal Article
LanguageEnglish
Published England BioMed Central Ltd 28.12.2011
BioMed Central
BMC
Subjects
Cancer
Deoxyribonucleic acid
Development and progression
DNA
DNA methylation
Genes
Genetic regulation
Genetics
genomics
Methylation
Physiological aspects
Polymerase chain reaction
Population studies
Studies
Technical Note
Canada
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Summary: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.
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ISSN:1756-0500
1756-0500
DOI:10.1186/1756-0500-4-565