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Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

Lookup NU author(s): Francesca Migheli, Professor John MathersORCiD

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

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.

Publication metadata

Author(s): Migheli F, Stoccoro A, Coppede F, Omar WAW, Failli A, Consolini R, Seccia M, Spisni R, Miccoli P, Mathers JC, Migliore L

Publication type: Article

Publication status: Published

Journal: PLoS One

Year: 2013

Volume: 8

Issue: 1

Print publication date: 11/01/2013

Date deposited: 09/07/2013

ISSN (electronic): 1932-6203

Publisher: Public Library of Science

URL: http://dx.doi.org/10.1371/journal.pone.0052501

DOI: 10.1371/journal.pone.0052501

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