title: A methylation-specific dot blot assay for improving specificity and sensitivity of methylation-specific PCR on DNA methylation analysis.
abstract: OBJECTIVE
  Developing a methylation-specific dot blot assay (MSP-DB) to increase the sensitivity and specificity of simultaneous methylation analysis of multiple genes is the goal of the present study to evaluate the methylation status of GSTP1 and RASSF1A from prostate cancer in Vietnamese males.
  
  
  METHODS
  The methylation of GSTP1 and RASSF1A was investigated by using the MSP in 50 prostate cancer and 17 benign prostate hyperplasia specimens. The MSP-DB assay that uses a single or multiple probes specifically detected the methylation status of a particular gene or of the two genes GSTP1 and RASSF1A at the same time in a series of samples. The sensitivity and specificity of the MSP-DB were compared to those of the MSP.
  
  
  RESULTS
  The probes specifically hybridized with the methylated targets only in the MSP-DB, which allowed detecting GSTP1 and RASSF1A methylation in 23 of 50 and 14 of 50 patients with prostate cancer and in 2 of 17 and 4 of 17 patients with benign prostate hyperplasia. MSP-DB following the MSP assay improved the sensitivity of detection to more than 0.01 % methylated status of a given high CpG-rich region. One methylated MSP product corresponding to the GSTP1, lack of methylated cytosine, was clearly detected on gel electrophoresis but barely visible on MSP-DB. Thus, the MSP-DB is suitable to eliminate the risk of false-positive results.
  
  
  CONCLUSIONS
  The MSP-DB dispels the weakness of MSP and increases the sensitivity to simultaneous methylation analysis of multiple genes. The MSP-DB is advantageous for the promotion of DNA methylation markers in progressing quickly toward clinical application.
authors.affiliation: Faculty of Biology, VNU University of Science, 334, Nguyen Trai, Thanh Xuan, Hanoi, Vietnam, vothithuonglan@hus.edu.vn.
authors.forename: Vo Thi Thuong
authors.initials: VT
authors.lastname: Lan
authors.forename: Nguyen Thu
authors.initials: NT
authors.lastname: Trang
authors.forename: Doan Thi Hong
authors.initials: DT
authors.lastname: Van
authors.forename: Ta Bich
authors.initials: TB
authors.lastname: Thuan
authors.forename: Ta
authors.initials: T
authors.lastname: Van To
authors.forename: Vuong Dieu
authors.initials: VD
authors.lastname: Linh
authors.forename: Nguyen Quynh
authors.initials: NQ
authors.lastname: Uyen
mesh_terms: D044466:Asian People; D019175:DNA Methylation; D051549:Glutathione S-Transferase pi / Q000235:genetics*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011470:Prostatic Hyperplasia / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000601:surgery; D012680:Sensitivity and Specificity; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi