Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis

Abstract

We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2’-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive and sensitive screening tool for identifying and characterizing DNA methylation changes to L1 elements at multiple regions across the genome.

Keywords: :

• DNA Methylation
• heterogeneous methylation
• high resolution melt analysis
• LINE1
• mouse tissue methylation

Acknowledgments

We thank Ami–Louise Cochrane, Mark Lawrence, Alex Staudacher and Katrina Bexis for assistance with animal procedures; Dr. Tanya Day for the 5-aza cell culture experiments; and Dr. Tina Bianco-Miotto for critically reading this manuscript. This work was supported by the Low Dose Radiation Research Program, Biological and Environmental Research, US. Department of Energy (DE-FG02–05ER64104); and the Flinders Medical Centre Foundation and BHP Billiton Low Dose Radiation Research Scholarship (to M.N.).