Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues
| Autor: | José de Assis Silva Júnior, Danielle Resende Camisasca, Débora dos Santos Tavares, Paulo Antônio Silvestre de Faria, Fernando Luiz Dias, Georgina Severo Ribeiro, Lídia Maria da Fonte de Amorim, Simone de Queiroz Chaves Lourenço |
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| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: | |
| Zdroj: | Jornal Brasileiro de Patologia e Medicina Laboratorial, Vol 50, Iss 2, Pp 150-158 (2014) |
| Druh dokumentu: | article |
| ISSN: | 1678-4774 1676-2444 |
| DOI: | 10.5935/1676-2444.20140009 |
| Popis: | Introduction:The tumor protein p53 gene (TP53) is a constant target of investigation in cancer pathogenesis. Analysis by immunohistochemistry provides limited data about p53 in oral carcinogenesis, and TP53sequencing can contribute to this analysis. However, obtaining high-quality and contamination-free deoxyribonucleic acid (DNA) for a proper amplification can be a difficult task when using paraffin-embedded tissues.Objective:Standardize DNA extraction, polymerase chain reaction (PCR) amplification and DNA sequencing techniques for TP53 mutation analysis.Material and methods:Thirty-nine cases of oral squamous cell carcinoma (OSCC) were selected from the Pathology Division of Instituto Nacional de Câncer (Inca). The DNA extraction method used was the QIAamp® DNA minikit® system. After DNA quantification by spectrophotometry, 250 ng of genetic material obtained from TP53 gene were amplified by PCR for exon 2 and by nested PCR for exon 6. Out of the total sample, 11 cases were selected for exon 2 sequencing. Results: The DNA samples presented mean concentration of 119.74 ± 88.86 ng/µl (28.9-556.4) and purity of 1.69 ± 0.18 (1-1.9). Thirty-three (84.6%) samples were amplified for exon 2, and all samples for exon 6 (39/100%). Readable sequencing data were obtained in 10 (90.9%) cases.Conclusion:Optimization of conditions for TP53 sequencing was obtained, and this will facilitate the analysis of mutations in paraffin-embedded tissues, allowing molecular retrospective studies. |
| Databáze: | Directory of Open Access Journals |
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