Background: We present two melting curve analysis (MCA)-based semiquantitative real time PCR techniques to detect the promoter methylation status of genes. The first, MCA-MSP, follows the same principle as standard MSP but it is performed in a real time thermalcycler with results being visualized in a melting curve. The second, MCA-Meth, uses a single pair of primers designed with no CpGs in its sequence. These primers amplify both unmethylated and methylated sequences. In clinical applications the MSP technique has revolutionized methylation detection by simplifying the analysis to a PCR-based protocol. MCA-analysis based techniques may be able to further improve and simplify methylation analyses by reducing starting DNA amounts, by introducing an all-in-one tube reaction and by eliminating a final gel stage for visualization of the result. The current study aimed at investigating the feasibility of both MCA-MSP and MCA-Meth in the analysis of promoter methylation, and at defining potential advantages and shortcomings in comparison to currently implemented techniques, i.e. bisulfite sequencing and standard MSP.

Methylation of CpG islands in gene promoters can lead to gene silencing. Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes. RASSF1A (3p21.3), NORE1A (1q32.1) and BLU (3p21.3) have been shown to be downregulated by methylation in cancer, and PTEN (10q23.3) and MGMT (10q26.1) are located in areas commonly deleted in astrocytomas. MGMT methylation predicts a better response and a longer overall survival in patients with glioblastomas treated with temozolomide. We analyzed 53 astrocytoma samples and 10 high-grade glioma cell lines. Gene expression was assessed by RT-PCR. Bisulfite sequencing, MSP and a melting curve analysis-based real-time PCR were performed to detect promoter methylation. Treatments with 5'-aza-2'-deoxicitidine were applied to restore gene expression in cell lines. Ninety-two percent of tumor samples were methylated for RASSF1A, 30%-57% for BLU and 47% for MGMT, suggesting promoter methylation of these genes to be a common event in glioma tumorigenesis. Only 4% of the tumors revealed a methylated promoter for NORE1A. No association between methylation and loss of expression could be established for PTEN. We identified de novo DNMTs overexpression in a subset of tumors which may explain the methylation phenotype of individual gliomas.