Promoter methylation in the PTCH gene in cervical epithelial cancer and ovarian cancer tissue as studied by eight novel Pyrosequencing® assays
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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.
Originalsprog | Engelsk |
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Tidsskrift | International Journal of Oncology Research |
Vol/bind | 38 |
Udgave nummer | 3 |
Sider (fra-til) | 685-92 |
Antal sider | 8 |
DOI | |
Status | Udgivet - mar. 2011 |
Eksternt udgivet | Ja |
ID: 46848160