title: Comparison study of MS-HRM and pyrosequencing techniques for quantification of APC and CDKN2A gene methylation.
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.
authors: Department of Translational Research and New Technologies in Medicine and Surgery, Division of Medical Genetics, University of Pisa, Pisa, Italy.
authors: Francesca
authors: F
authors: Migheli
authors: Andrea
authors: A
authors: Stoccoro
authors: Fabio
authors: F
authors: Coppedè
authors: Wan Adnan
authors: WA
authors: Wan Omar
authors: Alessandra
authors: A
authors: Failli
authors: Rita
authors: R
authors: Consolini
authors: Massimo
authors: M
authors: Seccia
authors: Roberto
authors: R
authors: Spisni
authors: Paolo
authors: P
authors: Miccoli
authors: John C
authors: JC
authors: Mathers
authors: Lucia
authors: L
authors: Migliore
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics*; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm