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.

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

Andrea Stoccoro;COPPEDE', FABIO;FAILLI, ALESSANDRA;CONSOLINI, RITA;SPISNI, ROBERTO;
2013-01-01

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.
2013
Francesca, Migheli; Stoccoro, Andrea; Coppede', Fabio; Wan Adnan Wan Omar, ; Failli, Alessandra; Consolini, Rita; Massimo, Seccia; Spisni, Roberto; Paolo, Miccoli; Mathers, John C.; Lucia, Migliore
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/686086
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