title: A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples.
abstract: BACKGROUND
  CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.
  
  
  METHODS
  We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.
  
  
  RESULTS
  The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.
  
  
  CONCLUSIONS
  The developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, 15771, Greece.
authors.forename: Lampros
authors.initials: L
authors.lastname: Dimitrakopoulos
authors.forename: Panagiotis A
authors.initials: PA
authors.lastname: Vorkas
authors.forename: Vasilis
authors.initials: V
authors.lastname: Georgoulias
authors.forename: Evi S
authors.initials: ES
authors.lastname: Lianidou
mesh_terms: D001483:Base Sequence; D001943:Breast Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055332:Cystatin M / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005260:Female; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
chemical_list: C525104:CST6 protein, human; D055332:Cystatin M; D004273:DNA, Neoplasm