Promoter methylation in the PTCH gene in cervical epithelial cancer and ovarian cancer tissue as studied by eight novel Pyrosequencing® assays
Research output: Contribution to journal › Journal article › Research › peer-review
DNA methylation status in the CpG sites of promoter regions in cancer-related genes, such as PTCH, has traditionally been investigated using either dye-terminator sequencing or methylation-specific PCR. We aimed to study the PTCH gene promoter methylation in gynecological cancers, with a method that gives a quantitative measure of the methylation status of the promoter region of the studied gene, and for this purpose, we designed novel Pyrosequencing-based assays. Bisulfite-treated genomic DNA (bsDNA) was amplified by standard PCR and applied to novel Pyrosequencing® assays, in order to measure the methylated fraction (%) at each CpG site of the PTCH gene promoter. We analyzed 22 squamous cell cervical cancer tissue specimens (11 with good and 11 with poor outcomes after radiotherapy) and 5 ovarian cancer tissue specimens matched with 5 normal ovarian tissue specimens. Six optimized PCR protocols which generated 8 Pyrosequencing assays covering 63 CpG sites in the promoter regions 1 and 2 as well as the previously unanalyzed promoter region 3 in the PTCH gene were developed. The 27 tumor tissue specimens and 5 normal tissues did not show any methylation within any of the 63 CpG sites. Our data suggest that methylation of the PTCH promoter is not a high-prevalence feature of squamous cell cervical cancer or ovarian cancer, but Pyrosequencing assays are a good method for studying promoter methylation.
Original language | English |
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Journal | International Journal of Oncology Research |
Volume | 38 |
Issue number | 3 |
Pages (from-to) | 685-92 |
Number of pages | 8 |
DOIs | |
Publication status | Published - Mar 2011 |
Externally published | Yes |
- Base Sequence, CpG Islands, DNA Methylation, Female, Gene Expression Regulation, Neoplastic, Gene Frequency, High-Throughput Nucleotide Sequencing, Humans, Matched-Pair Analysis, Molecular Sequence Data, Neoplasms, Glandular and Epithelial, Ovarian Neoplasms, Promoter Regions, Genetic, Receptors, Cell Surface, Sequence Analysis, DNA, Uterine Cervical Neoplasms
Research areas
ID: 46848160