A methylation-specific dot blot assay for improving specificity and sensitivity of methylation-specific PCR on DNA methylation analysis.

BACKGROUND AND OBJECTIVES: 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. CONCLUSION: 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.

Standardization of the methylation­specific PCR method for analyzing BRCA1 and ER methylation.

The significant differences in DNA methylation that are considered to be a biomarker for the diagnosis of cancer are a barrier to the application of biomarkers in the clinical field. In the present study, new primers were designed and further standard controls were set up to validate the accuracy of the methylation­specific PCR (MSP), a method widely used to analyze DNA methylation. By analyzing the methylation pattern of breast cancer 1 (BRCA1) and estrogen receptor (ER) in 60 patients with breast cancer, the number of cases of methylated BRCA1 and ER detected by the primer was 7/60 and 21/60, respectively, whereas that detected by the previous widely used primers was 25/60 and 47/60, respectively. Sequencing of the MSP products indicated that the 18 and 26 false-positive methylations of BRCA1 and ER, respectively, were due to insufficient validation of the previously used primers. Thus, the present study proposes that all studies based on the MSP approach should incorporate more controls to validate the precision of the MSP primers.

Methylation profile of BRCA1, RASSF1A and ER in Vietnamese women with ovarian cancer.

DNA methylation is considered a promising biomarkers for diagnosis of cancer in general and of ovarian cancer in particular. In our study, we validated the accuracy of methylation specific polymerase chain reaction (MSP) to analyze the methylation pattern of BRCA1, RASSF1A and ER in 59 and 10 Vietnamese patients with epithelial ovarian cancer (EOC) and benign ovarian tumors, respectively. We found methylation of BRCA1, RASSF1A and ER in 11/59 (18.6%), 40/59 (67.8%) and 15/59 (25.4%) of EOC cases, while methylation of BRCA1 was only detected in 2/10 (20%) benign ovarian patients. Forty five out of the 59 EOCs (78%) demonstrated methylation at one or more genes. The methylation frequency of RASSF1A was significantly associated with EOC (p<0.0005). No significant association was observed between methylation status of these genes and the clinical and pathological parameters of tumors collected from Vietnamese women suffering from ovarian cancer.