Comparison study of MS-HRM and pyrosequencing techniques for quantification of APC and CDKN2A gene methylation

• Published in: PloS one Vol. 8; no. 1; p. e52501
• Main Authors: Migheli, Francesca, Stoccoro, Andrea, Coppedè, Fabio, Wan Omar, Wan Adnan, Failli, Alessandra, Consolini, Rita, Seccia, Massimo, Spisni, Roberto, Miccoli, Paolo, Mathers, John C, Migliore, Lucia
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11.01.2013; Public Library of Science (PLoS)
• Subjects: Adenomatous polyposis coli; Adenomatous Polyposis Coli Protein - genetics; Aging; Base Sequence; Biology; Biomarkers; Biomedical research; CDKN2A gene; Cell cycle; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - genetics; Comparative analysis; Correlation coefficient; Correlation coefficients; Cost analysis; CpG Islands - genetics; Cyclin-Dependent Kinase Inhibitor p16 - genetics; Deoxyribonucleic acid; DNA; DNA Methylation; DNA sequencing; DNA, Neoplasm - analysis; DNA, Neoplasm - genetics; Epigenetics; Gene expression; Hospitals; Humans; Interpolation; Laboratories; Medical prognosis; Medicine; Methods; Methylation; Molecular Sequence Data; Nucleic Acid Denaturation; Nutrition research; Pathology; Promoter Regions, Genetic - genetics; Reproducibility of Results; Sequence Analysis, DNA - methods; Sodium; Surgery; Temperature; Tissues; Tumorigenesis; Italy; Malaysia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0052501

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing.