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

Xia, Peng and Radpour, Ramin and Zachariah, Rebecca and Fan, Alex Xiu Cheng and Kohler, Corina and Hahn, Sinuhe and Holzgreve, Wolfgang and Zhong, Xiao Yan. (2009) Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR. International journal of genetics and molecular biology, Vol. 32. pp. 20-24.

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Official URL: http://edoc.unibas.ch/dok/A6007032

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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.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Former Units at DBM > Gynecological Oncology (Zhong)
UniBasel Contributors:Zhong, Xiao Yan
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Academic Journals
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:26 Apr 2013 06:59
Deposited On:01 Mar 2013 11:11

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