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dc.creatorLorente, Aialaes_ES
dc.creatorMueller, Wolfes_ES
dc.creatorUrdangarín, Edurnees_ES
dc.creatorLázcoz, Paulaes_ES
dc.creatorDeimling, Andreas vones_ES
dc.creatorSáez Castresana, Javieres_ES
dc.date.accessioned2014-06-04T11:15:17Z
dc.date.available2014-06-04T11:15:17Z
dc.date.issued2008
dc.identifier.issn1471-2407
dc.identifier.other767
dc.identifier.urihttps://hdl.handle.net/2454/10728
dc.description.abstractBackground: 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. Methods: The promoters of the RASSF1A (3p21.3), BLU (3p21.3) and MGMT (10q26) genes were analyzed by MCA-MSP and MCA-Meth in 13 astrocytoma samples, 6 high grade glioma cell lines and 4 neuroblastoma cell lines. The data were compared with standard MSP and validated by bisulfite sequencing. Results: Both, MCA-MSP and MCA-Meth, successfully determined promoter methylation. MCA-MSP provided information similar to standard MSP analyses. However the analysis was possible in a single tube and avoided the gel stage. MCA-Meth proved to be useful in samples with intermediate methylation status, reflected by a melting curve position shift in dependence on methylation extent. Conclusion: We propose MCA-MSP and MCA-Meth as alternative or supplementary techniques to MSP or bisulfite sequencing.en
dc.description.sponsorshipThis research was supported in part by grants from the Departments of Health and Education of the Government of Navarra, Pamplona; Fondo de Investigación Sanitaria (PI031356), and Ministerio de Ciencia y Tecnología y Fondo Europeo de Desarrollo Regional (BFI2003-08775), Madrid, Spain.en
dc.format.mimetypeapplication/pdfen
dc.language.isoengen
dc.publisherBioMed Centralen
dc.relation.ispartofBMC Cancer, 2008, 8: 61en
dc.rights© 2008 Lorente et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.en
dc.rights.urihttp://creativecommons.org/licenses/by/2.0/
dc.subjectTumor suppressor geneen
dc.subjectPolymerase chain reactionen
dc.subjectEpigeneticen
dc.subjectInactivationen
dc.subjectO-6-Methylguanine DNA methyltraspheraseen
dc.subjectHypermethylationen
dc.subjectRASSF1Aen
dc.subjectThreeP21.3 [3P21.3]en
dc.subjectCanceren
dc.subjectProtocolen
dc.subjectAssayen
dc.subjectOncologyen
dc.titleDetection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCRen
dc.typeArtículo / Artikuluaes
dc.typeinfo:eu-repo/semantics/articleen
dc.contributor.departmentUniversidad Pública de Navarra. Departamento de Ciencias de la Saludes_ES
dc.contributor.departmentNafarroako Unibertsitate Publikoa. Osasun Zientziak Sailaeu
dc.rights.accessRightsAcceso abierto / Sarbide irekiaes
dc.rights.accessRightsinfo:eu-repo/semantics/openAccessen
dc.identifier.doi10.1186/1471-2407-8-61
dc.relation.publisherversionhttps://dx.doi.org/10.1186/1471-2407-8-61
dc.type.versionVersión publicada / Argitaratu den bertsioaes
dc.type.versioninfo:eu-repo/semantics/publishedVersionen


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© 2008 Lorente et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Except where otherwise noted, this item's license is described as © 2008 Lorente et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.