PML-RARalpha gene detection method optimization for quantitative PCR
Autor(a) principal: | |
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Data de Publicação: | 2008 |
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=S1676-24442008000100003 |
Resumo: | Hybrid gene PML-RARalpha is the molecular target found in most cases of acute promyelocytic leukemia (APL) and has been used for diagnosis and minimal residual disease studies. The standard molecular technique employed is qualitative reverse transcriptase-polymerase chain reaction (RT-PCR), but with the emergence of real time PCR (Q-PCR), PML-RARalpha gene detection approaches have been described allowing transcript detection, with the methodological advantage of eliminating post-PCR processing. However, current protocols report the use of expensive fluorescent labeled probes, limiting its routine application in the laboratory. The objective of this study was to optimize PML-RARalpha gene detection method for Q-PCR, using SYBR® Green fluorescent dye. The analysis was performed with NB4 cellular lineage cDNA. Thermal cycling protocols, cDNA synthesis with random or specific primer and different MgCl2 and amplification primers concentrations were tested. Results show that amplification improved in the following conditions: 2 mM MgCl2, 10 pmol primers and cDNA synthesized with specific primer. There were no significant differences using annealing temperature (58°C/30 s) followed by extension (72°C/30 s) or annealing associated with extension as a single step (60°C/45 s). This paper demonstrates the optimization of PML-RARalpha gene detection for Q-PCR studies using a technique considered sensitive and less expensive for routine use in the laboratory. |
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PML-RARalpha gene detection method optimization for quantitative PCRAPLPML-RARalphaQ-PCRSYBR® GreenHybrid gene PML-RARalpha is the molecular target found in most cases of acute promyelocytic leukemia (APL) and has been used for diagnosis and minimal residual disease studies. The standard molecular technique employed is qualitative reverse transcriptase-polymerase chain reaction (RT-PCR), but with the emergence of real time PCR (Q-PCR), PML-RARalpha gene detection approaches have been described allowing transcript detection, with the methodological advantage of eliminating post-PCR processing. However, current protocols report the use of expensive fluorescent labeled probes, limiting its routine application in the laboratory. The objective of this study was to optimize PML-RARalpha gene detection method for Q-PCR, using SYBR® Green fluorescent dye. The analysis was performed with NB4 cellular lineage cDNA. Thermal cycling protocols, cDNA synthesis with random or specific primer and different MgCl2 and amplification primers concentrations were tested. Results show that amplification improved in the following conditions: 2 mM MgCl2, 10 pmol primers and cDNA synthesized with specific primer. There were no significant differences using annealing temperature (58°C/30 s) followed by extension (72°C/30 s) or annealing associated with extension as a single step (60°C/45 s). This paper demonstrates the optimization of PML-RARalpha gene detection for Q-PCR studies using a technique considered sensitive and less expensive for routine use in the laboratory.Sociedade Brasileira de Patologia Clínica2008-02-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442008000100003Jornal Brasileiro de Patologia e Medicina Laboratorial v.44 n.1 2008reponame:Jornal Brasileiro de Patologia e Medicina Laboratorial (Online)instname:Sociedade Brasileira de Patologia (SBP)instacron:SBP10.1590/S1676-24442008000100003info:eu-repo/semantics/openAccessVasconcellos,Jaíra Ferreira deMelo,Raul Antônio MoraisMelo,Fárida Coeli Barros CorreiaNeves,Washington BatistaKido,Éderson Akioeng2008-05-15T00:00:00Zoai:scielo:S1676-24442008000100003Revistahttp://www.scielo.br/jbpmlhttps://old.scielo.br/oai/scielo-oai.php||jbpml@sbpc.org.br1678-47741676-2444opendoar:2008-05-15T00:00Jornal Brasileiro de Patologia e Medicina Laboratorial (Online) - Sociedade Brasileira de Patologia (SBP)false |
dc.title.none.fl_str_mv |
PML-RARalpha gene detection method optimization for quantitative PCR |
title |
PML-RARalpha gene detection method optimization for quantitative PCR |
spellingShingle |
PML-RARalpha gene detection method optimization for quantitative PCR Vasconcellos,Jaíra Ferreira de APL PML-RARalpha Q-PCR SYBR® Green |
title_short |
PML-RARalpha gene detection method optimization for quantitative PCR |
title_full |
PML-RARalpha gene detection method optimization for quantitative PCR |
title_fullStr |
PML-RARalpha gene detection method optimization for quantitative PCR |
title_full_unstemmed |
PML-RARalpha gene detection method optimization for quantitative PCR |
title_sort |
PML-RARalpha gene detection method optimization for quantitative PCR |
author |
Vasconcellos,Jaíra Ferreira de |
author_facet |
Vasconcellos,Jaíra Ferreira de Melo,Raul Antônio Morais Melo,Fárida Coeli Barros Correia Neves,Washington Batista Kido,Éderson Akio |
author_role |
author |
author2 |
Melo,Raul Antônio Morais Melo,Fárida Coeli Barros Correia Neves,Washington Batista Kido,Éderson Akio |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
Vasconcellos,Jaíra Ferreira de Melo,Raul Antônio Morais Melo,Fárida Coeli Barros Correia Neves,Washington Batista Kido,Éderson Akio |
dc.subject.por.fl_str_mv |
APL PML-RARalpha Q-PCR SYBR® Green |
topic |
APL PML-RARalpha Q-PCR SYBR® Green |
description |
Hybrid gene PML-RARalpha is the molecular target found in most cases of acute promyelocytic leukemia (APL) and has been used for diagnosis and minimal residual disease studies. The standard molecular technique employed is qualitative reverse transcriptase-polymerase chain reaction (RT-PCR), but with the emergence of real time PCR (Q-PCR), PML-RARalpha gene detection approaches have been described allowing transcript detection, with the methodological advantage of eliminating post-PCR processing. However, current protocols report the use of expensive fluorescent labeled probes, limiting its routine application in the laboratory. The objective of this study was to optimize PML-RARalpha gene detection method for Q-PCR, using SYBR® Green fluorescent dye. The analysis was performed with NB4 cellular lineage cDNA. Thermal cycling protocols, cDNA synthesis with random or specific primer and different MgCl2 and amplification primers concentrations were tested. Results show that amplification improved in the following conditions: 2 mM MgCl2, 10 pmol primers and cDNA synthesized with specific primer. There were no significant differences using annealing temperature (58°C/30 s) followed by extension (72°C/30 s) or annealing associated with extension as a single step (60°C/45 s). This paper demonstrates the optimization of PML-RARalpha gene detection for Q-PCR studies using a technique considered sensitive and less expensive for routine use in the laboratory. |
publishDate |
2008 |
dc.date.none.fl_str_mv |
2008-02-01 |
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=S1676-24442008000100003 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1676-24442008000100003 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/S1676-24442008000100003 |
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.44 n.1 2008 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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1752122294468083712 |