Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

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ID: 65792
2009
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Abstract
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.
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xia2009simultaneousgenetics Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Xia, Peng;Radpour, Ramin;Zachariah, Rebecca;Fan, Alex Xiu Cheng;Kohler, Corina;Hahn, Sinuhe;Holzgreve, Wolfgang;Zhong, Xiao Yan;
Journal Genetics and molecular biology
Year 2009
DOI
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URL
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572009000100003
Keywords
Medicine (General) Medicine Science Immunologic diseases. Allergy public aspects of medicine arctic medicine. tropical medicine genetics

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