title: Novel multiplex MethyLight protocol for detection of DNA methylation in patient tissues and bodily fluids.
abstract: Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings.
authors.affiliation: 1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada [2] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Olkhov-Mitsel
authors.affiliation: 1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada [2] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Darko
authors.initials: D
authors.lastname: Zdravic
authors.affiliation: Ontario Cancer Institute, Princess Margaret Cancer Center-University Health Network, Toronto, ON, Canada.
authors.forename: Ken
authors.initials: K
authors.lastname: Kron
authors.affiliation: 1] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada [2] Department of Pathology and Laboratory Medicine, University Health Network, Toronto, Ontario, Canada.
authors.forename: Theodorus
authors.initials: T
authors.lastname: van der Kwast
authors.affiliation: Department of Urology, University Health Network, Toronto, ON, Canada.
authors.forename: Neil
authors.initials: N
authors.lastname: Fleshner
authors.affiliation: 1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada [2] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada [3] Department of Pathology and Laboratory Medicine, University Health Network, Toronto, Ontario, Canada.
authors.forename: Bharati
authors.initials: B
authors.lastname: Bapat
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics* / Q000652:urine; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm; D018398:Homeodomain Proteins / Q000235:genetics* / Q000652:urine; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000652:urine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014157:Transcription Factors; D053781:Transforming Growth Factor beta2 / Q000235:genetics* / Q000652:urine
chemical_list: C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; D017931:DNA Primers; D004273:DNA, Neoplasm; D018398:Homeodomain Proteins; C509041:TGFB2 protein, human; D014157:Transcription Factors; D053781:Transforming Growth Factor beta2; C073547:HOXA4 protein, human