Data/hora: 07/06/2024 04:48:48

Provedor de dados: | 74
País: | Brazil
Título: | Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR
Autores: | Xia,Peng; Radpour,Ramin; Zachariah,Rebecca; Fan,Alex Xiu Cheng; Kohler,Corina; Hahn,Sinuhe; Holzgreve,Wolfgang; Zhong,Xiao Yan.
Data: | 2009-01-01
Ano: | 2009
Palavras-chave: | Circulating cell-free DNA; Mitochondrial DNA; Nuclear DNA; Real-time PCR; Quantitative PCR.
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.
Tipo: | Info:eu-repo/semantics/article
Idioma: | Inglês
Identificador: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572009000100003
Editor: | Sociedade Brasileira de Genética
Relação: | 10.1590/S1415-47572009000100003
Formato: | text/html
Fonte: | Genetics and Molecular Biology  v.32 n.1 2009
Fonte: | info:eu-repo/semantics/openAccess