Extração de DNA para a análise da amelogenina em amostras fixadas em formalina, incluídas em parafina e arquivadas por 1 e 5 anos no Departamento de Patologia da Universidade Federal de São Paulo

• Ano de defesa: 2012
• Autor(a) principal: Funabashi, Karina Silva [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: extração de DNA; paraffin; parafina; Amelogenina; DNA extraction
• Link de acesso: http://repositorio.unifesp.br/handle/11600/9627

Formalin fixed and paraffin embedded tissues provide valuable retrospective investigations for molecular studies, especially for genetic studies, when the DNA is not available in fresh and/or frozen samples. However, according to some authors, is difficult to obtain a DNA of good quality, since the fixation process results in nucleic acids fragmentation. The aim of this study was to evaluate the DNA extracted from paraffin embedded tissues, after 1 and 5 years of storage, by 3 methods of extraction. For this, the gene of β-actin (136pb) were used, to detect the viability and DNA fragmentation, and the gene of amelogenin (X: 212pb e Y: 218pb) for sexual differentiation and viability in primers with greater length. The study involved 12 recent autopsy cases, where samples of liver (n=10), spleen (n=10) and brain (n=10) were collected in duplicate, which one group followed to the process of fixation and inclusion and the other group followed to freezing. Moreover, the same kind of tissues, normal (n=10 each), from 13 autopsy cases archived for 1 year and 15 cases archived for 5 years were used. After paraffin remove, the samples were submitted to DNA extraction with commercial kit (QIAGEN QIAamp Mini), Salting-Out and phenol-chlorophorm. The DNA extracted was quantified in Nanodrop® and adjusted for PCR (10ng/μl). The PCR products were visualized in agarose gel 1%. The samples of spleen and liver showed more yield in DNA extraction than the brain samples, in all the times. All the samples archived showed good extraction conditions, however should take in consideration the fixation and embedded process, which could compromise the DNA quality. The extraction by phenol-chloroform yielded more DNA quantity and purity than the other methods. However, the commercial kit extraction showed better results in DNA amplification. The primer of the gene of amelogenin was amplified in all utilized samples, however recommends the utilization of smaller primers for a complete analyze of the fragment studied.