Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR
Autor(a) principal: | |
---|---|
Data de Publicação: | 2009 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Genetics and Molecular Biology |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572009000100003 |
Resumo: | Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency. |
id |
SBG-1_b4b0eaa46eca7f70518b96b4173988ef |
---|---|
oai_identifier_str |
oai:scielo:S1415-47572009000100003 |
network_acronym_str |
SBG-1 |
network_name_str |
Genetics and Molecular Biology |
repository_id_str |
|
spelling |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCRcirculating cell-free DNAmitochondrial DNAnuclear DNAreal-time PCRquantitative PCRQuantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.Sociedade Brasileira de Genética2009-01-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572009000100003Genetics and Molecular Biology v.32 n.1 2009reponame:Genetics and Molecular Biologyinstname:Sociedade Brasileira de Genética (SBG)instacron:SBG10.1590/S1415-47572009000100003info:eu-repo/semantics/openAccessXia,PengRadpour,RaminZachariah,RebeccaFan,Alex Xiu ChengKohler,CorinaHahn,SinuheHolzgreve,WolfgangZhong,Xiao Yaneng2009-02-10T00:00:00Zoai:scielo:S1415-47572009000100003Revistahttp://www.gmb.org.br/ONGhttps://old.scielo.br/oai/scielo-oai.php||editor@gmb.org.br1678-46851415-4757opendoar:2009-02-10T00:00Genetics and Molecular Biology - Sociedade Brasileira de Genética (SBG)false |
dc.title.none.fl_str_mv |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR |
title |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR |
spellingShingle |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR Xia,Peng circulating cell-free DNA mitochondrial DNA nuclear DNA real-time PCR quantitative PCR |
title_short |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR |
title_full |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR |
title_fullStr |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR |
title_full_unstemmed |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR |
title_sort |
Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR |
author |
Xia,Peng |
author_facet |
Xia,Peng Radpour,Ramin Zachariah,Rebecca Fan,Alex Xiu Cheng Kohler,Corina Hahn,Sinuhe Holzgreve,Wolfgang Zhong,Xiao Yan |
author_role |
author |
author2 |
Radpour,Ramin Zachariah,Rebecca Fan,Alex Xiu Cheng Kohler,Corina Hahn,Sinuhe Holzgreve,Wolfgang Zhong,Xiao Yan |
author2_role |
author author author author author author author |
dc.contributor.author.fl_str_mv |
Xia,Peng Radpour,Ramin Zachariah,Rebecca Fan,Alex Xiu Cheng Kohler,Corina Hahn,Sinuhe Holzgreve,Wolfgang Zhong,Xiao Yan |
dc.subject.por.fl_str_mv |
circulating cell-free DNA mitochondrial DNA nuclear DNA real-time PCR quantitative PCR |
topic |
circulating cell-free DNA mitochondrial DNA nuclear DNA real-time PCR quantitative PCR |
description |
Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency. |
publishDate |
2009 |
dc.date.none.fl_str_mv |
2009-01-01 |
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=S1415-47572009000100003 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572009000100003 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/S1415-47572009000100003 |
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 Genética |
publisher.none.fl_str_mv |
Sociedade Brasileira de Genética |
dc.source.none.fl_str_mv |
Genetics and Molecular Biology v.32 n.1 2009 reponame:Genetics and Molecular Biology instname:Sociedade Brasileira de Genética (SBG) instacron:SBG |
instname_str |
Sociedade Brasileira de Genética (SBG) |
instacron_str |
SBG |
institution |
SBG |
reponame_str |
Genetics and Molecular Biology |
collection |
Genetics and Molecular Biology |
repository.name.fl_str_mv |
Genetics and Molecular Biology - Sociedade Brasileira de Genética (SBG) |
repository.mail.fl_str_mv |
||editor@gmb.org.br |
_version_ |
1752122381534494720 |