EpiHRMAssay, in tube and in silico combined approach for the scanning and epityping of heterogeneous DNA methylation

Marco Cirilli; Ines Delfino; Emilia Caboni; Rosario Muleo

doi:10.1093/biomethods/bpw008

EpiHRMAssay, in tube and in silico combined approach for the scanning and epityping of heterogeneous DNA methylation

Biol Methods Protoc. 2017 Jan 25;2(1):bpw008. doi: 10.1093/biomethods/bpw008. eCollection 2017 Jan.

Authors

Marco Cirilli¹, Ines Delfino², Emilia Caboni³, Rosario Muleo¹

Affiliations

¹ Department of Agricultural and Forestry Sciences, University of Tuscia, Via S. Camillo de Lellis snc, Viterbo 01100, Italy.
² Department of Ecological and Biological Sciences, University of Tuscia, L.go dell'Università snc, Viterbo 01100, Italy.
³ Council for Agricultural Research and Analysis of Agricultural Economics (CREA), Fruit Tree Research Center, Via di Fioranello, 52, 00134 Rome, Italy.

Abstract

Reliable and cost-effective assays with adequate sensitivity are required to detect the DNA methylation profile in plants for scientific and industrial purposes. The proposed novel assay, named EpiHRMAssay, allows to quantify the overall methylation status at target loci and to enable high-throughput analyses. It combines in tube High Resolution Melting Analysis on bisulphite-treated templates with the in silico prediction of the melting profile of virtual epialleles using uMELT^SM software. The predicted melting temperatures (T _m-s) of a set of epialleles characterized by different numbers of methylated cytosines (#^mC) or different ^mC configurations were obtained and used to build calibration models, enabling the quantification of methylation in unknown samples using only the in tube observed melting temperature (T _m-o). EpiHRMAssay was validated by analysing the promoter region of CMT3, DDM1, and ROS1 genes involved in the regulation of methylation/demethylation processes and chromatin remodelling within a population of peach plants. Results demonstrate that EpiHRMAssay is a sensitive and reliable tool for locus-specific large-scale research and diagnostic contexts of the regulative regions of genes, in a broad range of organisms, including mammals. EpiHRMAssay also provides complementary information for the assessment of heterogeneous methylation and can address an array of biological questions on epigenetic regulation for diversity studies and for large-scale functional genomics.

Keywords: DNA methylation; HRMA; High Resolution Melting Analysis; epigenetic regulation; in silico melting prediction; somaclonal variation.