Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Jornal Brasileiro de Patologia e Medicina Laboratorial (Online) |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442014000200150 |
Resumo: | 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. |
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Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissuesTP53 genesquamous cell carcinomaoral cancerDNA sequencingmethodologyIntroduction: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.Sociedade Brasileira de Patologia Clínica2014-04-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442014000200150Jornal Brasileiro de Patologia e Medicina Laboratorial v.50 n.2 2014reponame:Jornal Brasileiro de Patologia e Medicina Laboratorial (Online)instname:Sociedade Brasileira de Patologia (SBP)instacron:SBP10.5935/1676-2444.20140009info:eu-repo/semantics/openAccessSilva Júnior,José de AssisCamisasca,Danielle ResendeTavares,Débora dos SantosFaria,Paulo Antônio Silvestre deDias,Fernando LuizRibeiro,Georgina SeveroAmorim,Lídia Maria da Fonte deLourenço,Simone de Queiroz Chaveseng2015-10-26T00:00:00Zoai:scielo:S1676-24442014000200150Revistahttp://www.scielo.br/jbpmlhttps://old.scielo.br/oai/scielo-oai.php||jbpml@sbpc.org.br1678-47741676-2444opendoar:2015-10-26T00:00Jornal Brasileiro de Patologia e Medicina Laboratorial (Online) - Sociedade Brasileira de Patologia (SBP)false |
dc.title.none.fl_str_mv |
Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues |
title |
Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues |
spellingShingle |
Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues Silva Júnior,José de Assis TP53 gene squamous cell carcinoma oral cancer DNA sequencing methodology |
title_short |
Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues |
title_full |
Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues |
title_fullStr |
Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues |
title_full_unstemmed |
Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues |
title_sort |
Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues |
author |
Silva Júnior,José de Assis |
author_facet |
Silva Júnior,José de Assis Camisasca,Danielle Resende Tavares,Débora dos Santos Faria,Paulo Antônio Silvestre de Dias,Fernando Luiz Ribeiro,Georgina Severo Amorim,Lídia Maria da Fonte de Lourenço,Simone de Queiroz Chaves |
author_role |
author |
author2 |
Camisasca,Danielle Resende Tavares,Débora dos Santos Faria,Paulo Antônio Silvestre de Dias,Fernando Luiz Ribeiro,Georgina Severo Amorim,Lídia Maria da Fonte de Lourenço,Simone de Queiroz Chaves |
author2_role |
author author author author author author author |
dc.contributor.author.fl_str_mv |
Silva Júnior,José de Assis Camisasca,Danielle Resende Tavares,Débora dos Santos Faria,Paulo Antônio Silvestre de Dias,Fernando Luiz Ribeiro,Georgina Severo Amorim,Lídia Maria da Fonte de Lourenço,Simone de Queiroz Chaves |
dc.subject.por.fl_str_mv |
TP53 gene squamous cell carcinoma oral cancer DNA sequencing methodology |
topic |
TP53 gene squamous cell carcinoma oral cancer DNA sequencing methodology |
description |
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. |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014-04-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442014000200150 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442014000200150 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.5935/1676-2444.20140009 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Sociedade Brasileira de Patologia Clínica |
publisher.none.fl_str_mv |
Sociedade Brasileira de Patologia Clínica |
dc.source.none.fl_str_mv |
Jornal Brasileiro de Patologia e Medicina Laboratorial v.50 n.2 2014 reponame:Jornal Brasileiro de Patologia e Medicina Laboratorial (Online) instname:Sociedade Brasileira de Patologia (SBP) instacron:SBP |
instname_str |
Sociedade Brasileira de Patologia (SBP) |
instacron_str |
SBP |
institution |
SBP |
reponame_str |
Jornal Brasileiro de Patologia e Medicina Laboratorial (Online) |
collection |
Jornal Brasileiro de Patologia e Medicina Laboratorial (Online) |
repository.name.fl_str_mv |
Jornal Brasileiro de Patologia e Medicina Laboratorial (Online) - Sociedade Brasileira de Patologia (SBP) |
repository.mail.fl_str_mv |
||jbpml@sbpc.org.br |
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1752122295922458624 |