title: Comparative analysis of methods to determine DNA methylation levels of a tumor-related microRNA gene.
abstract: Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. In the current study, we compared three methods commonly used for quantifying DNA methylation-bisulfite pyrosequencing, quantitative methylation-specific PCR (Q-MSP), and MethyLight-by focusing on the CpG island of the gene encoding the microRNA-34b and microRNA-34c (miR-34b/c); aberrant regulation of this miR is associated with various human malignancies, including gastric cancer. Standard curve analysis using control DNA samples demonstrated the highest quantitative accuracy in Q-MSP analysis. We also carried out methylation analysis using gastric mucosa specimens obtained from gastric cancer patients. We found a high correlation between methylation levels determined by Q-MSP and those determined by MethyLight (R(2)=0.952), whereas the results of bisulfite pyrosequencing and the other two methods were less well correlated (R(2)=0.864 and R(2)=0.804 for Q-MSP and MethyLight, respectively). This may reflect possible PCR bias in the pyrosequencing technique, which we show can be corrected for by applying a cubic approximate equation to the original data. Thus, although results obtained by the different DNA methylation analysis techniques are largely comparable, an appropriate correction may be necessary for stringent comparison.
authors.affiliation: Research & Development Department, SRL, Tokyo 191-0002, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Konishi
authors.affiliation: Research & Development Department, SRL, Tokyo 191-0002, Japan. Electronic address: hayash@srl.srl-inc.co.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hayashi
authors.affiliation: Department of Molecular Biology, Sapporo Medical University, Sapporo 060-8556, Japan.
authors.forename: Hiromu
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Molecular Biology, Sapporo Medical University, Sapporo 060-8556, Japan.
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Yamamoto
authors.affiliation: Research & Development Department, SRL, Tokyo 191-0002, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Sugisaki
authors.affiliation: Research & Development Department, SRL, Tokyo 191-0002, Japan. Electronic address: higashi@srl.srl-inc.co.jp.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Higashimoto
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013274:Stomach Neoplasms / Q000175:diagnosis / Q000235:genetics*; D013447:Sulfites / Q000494:pharmacology
chemical_list: C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D013447:Sulfites; C042345:hydrogen sulfite
keywords: MSP; Methylation; Pyrosequencing; miR-34b/c