Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis
Resistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMT gene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMT promoter methylation plays an important role as...
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2007
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| Online Access: | http://hdl.handle.net/10725/6179 https://doi.org/10.2353/jmoldx.2007.060167 http://libraries.lau.edu.lb/research/laur/terms-of-use/articles.php http://www.sciencedirect.com/science/article/pii/S1525157810604060 |
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| _version_ | 1864513478639747072 |
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| author | El Maarri, Osman |
| author2 | Mikeska, Thomas Bock, Christoph Hubner, Anika Ehrentraut, Denise Schramm, Johannes Felsberg, Jorg Khl, Philip Buttner, Reinhard Pietsch, Torsten Waha, Andreas |
| author2_role | author author author author author author author author author author |
| author_facet | El Maarri, Osman Mikeska, Thomas Bock, Christoph Hubner, Anika Ehrentraut, Denise Schramm, Johannes Felsberg, Jorg Khl, Philip Buttner, Reinhard Pietsch, Torsten Waha, Andreas |
| author_role | author |
| dc.creator.none.fl_str_mv | El Maarri, Osman Mikeska, Thomas Bock, Christoph Hubner, Anika Ehrentraut, Denise Schramm, Johannes Felsberg, Jorg Khl, Philip Buttner, Reinhard Pietsch, Torsten Waha, Andreas |
| dc.date.none.fl_str_mv | 2007 2017-09-13T09:03:11Z 2017-09-13T09:03:11Z 2017-09-13 |
| dc.identifier.none.fl_str_mv | 1943-7811 http://hdl.handle.net/10725/6179 https://doi.org/10.2353/jmoldx.2007.060167 Mikeska, T., Bock, C., El-Maarri, O., Hübner, A., Ehrentraut, D., Schramm, J., ... & Waha, A. (2007). Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis. The Journal of Molecular Diagnostics, 9(3), 368-381. http://libraries.lau.edu.lb/research/laur/terms-of-use/articles.php http://www.sciencedirect.com/science/article/pii/S1525157810604060 |
| dc.language.none.fl_str_mv | en |
| dc.relation.none.fl_str_mv | The Journal of Molecular Diagnostics |
| dc.rights.*.fl_str_mv | info:eu-repo/semantics/openAccess |
| dc.title.none.fl_str_mv | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis |
| dc.type.none.fl_str_mv | Article info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/article |
| description | Resistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMT gene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMT promoter methylation plays an important role as a predictive biomarker for chemotherapy resistance. To adopt this established correlation into a molecular diagnosis procedure, we compared and optimized three experimental techniques [combined bisulfite restriction analysis, a primer extension- and denaturing high-performance liquid chromatography-based method named SIRPH (SNuPE ion pair-reverse phase high-performance liquid chromatography), and pyrosequencing] with regard to their accuracy of detecting MGMT promoter methylation. Initially, bisulfite sequencing was used to obtain a comprehensive methylation profile of the MGMT promoter region in 22 glioblastoma samples and in three normal brain controls. Next, we statistically identified CpG sites that best discriminate between methylated and unmethylated MGMT promoters. These results were then used to design optimal combined bisulfite restriction analysis, SIRPH, and pyrosequencing assays for accurate and cost-efficient assessment of MGMT promoter methylation. We compared all three techniques with regard to their reliability and reproducibility on well-characterized tumor samples. The optimized pyrosequencing assay performed best and provides a sensitive, robust, and easy-to-use method for quantitative assessment of MGMT methylation, for both snap-frozen and paraffin-embedded specimens. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | LAURepo_c9f2fb38791ef06427c2e992ded5aa4f |
| identifier_str_mv | 1943-7811 Mikeska, T., Bock, C., El-Maarri, O., Hübner, A., Ehrentraut, D., Schramm, J., ... & Waha, A. (2007). Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis. The Journal of Molecular Diagnostics, 9(3), 368-381. |
| language_invalid_str_mv | en |
| network_acronym_str | LAURepo |
| network_name_str | Lebanese American University repository |
| oai_identifier_str | oai:laur.lau.edu.lb:10725/6179 |
| publishDate | 2007 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| spelling | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysisEl Maarri, OsmanMikeska, ThomasBock, ChristophHubner, AnikaEhrentraut, DeniseSchramm, JohannesFelsberg, JorgKhl, PhilipButtner, ReinhardPietsch, TorstenWaha, AndreasResistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMT gene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMT promoter methylation plays an important role as a predictive biomarker for chemotherapy resistance. To adopt this established correlation into a molecular diagnosis procedure, we compared and optimized three experimental techniques [combined bisulfite restriction analysis, a primer extension- and denaturing high-performance liquid chromatography-based method named SIRPH (SNuPE ion pair-reverse phase high-performance liquid chromatography), and pyrosequencing] with regard to their accuracy of detecting MGMT promoter methylation. Initially, bisulfite sequencing was used to obtain a comprehensive methylation profile of the MGMT promoter region in 22 glioblastoma samples and in three normal brain controls. Next, we statistically identified CpG sites that best discriminate between methylated and unmethylated MGMT promoters. These results were then used to design optimal combined bisulfite restriction analysis, SIRPH, and pyrosequencing assays for accurate and cost-efficient assessment of MGMT promoter methylation. We compared all three techniques with regard to their reliability and reproducibility on well-characterized tumor samples. The optimized pyrosequencing assay performed best and provides a sensitive, robust, and easy-to-use method for quantitative assessment of MGMT methylation, for both snap-frozen and paraffin-embedded specimens.PublishedN/A2017-09-13T09:03:11Z2017-09-13T09:03:11Z20072017-09-13Articleinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article1943-7811http://hdl.handle.net/10725/6179https://doi.org/10.2353/jmoldx.2007.060167Mikeska, T., Bock, C., El-Maarri, O., Hübner, A., Ehrentraut, D., Schramm, J., ... & Waha, A. (2007). Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis. The Journal of Molecular Diagnostics, 9(3), 368-381.http://libraries.lau.edu.lb/research/laur/terms-of-use/articles.phphttp://www.sciencedirect.com/science/article/pii/S1525157810604060enThe Journal of Molecular Diagnosticsinfo:eu-repo/semantics/openAccessoai:laur.lau.edu.lb:10725/61792021-03-19T10:00:49Z |
| spellingShingle | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis El Maarri, Osman |
| status_str | publishedVersion |
| title | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis |
| title_full | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis |
| title_fullStr | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis |
| title_full_unstemmed | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis |
| title_short | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis |
| title_sort | Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis |
| url | http://hdl.handle.net/10725/6179 https://doi.org/10.2353/jmoldx.2007.060167 http://libraries.lau.edu.lb/research/laur/terms-of-use/articles.php http://www.sciencedirect.com/science/article/pii/S1525157810604060 |