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

Genetics and Molecular Biology

Peng Xia²  Ramin Radpour²  Rebecca Zachariah²  Alex Xiu Cheng Fan²  Corina Kohler²  Sinuhe Hahn²  Wolfgang Holzgreve¹  Xiao Yan Zhong² 

[1] ,University of Basel Women's Hospital Laboratory for Prenatal Medicine and Gynaecological Oncology,Switzerland

关键词: circulating cell-free DNA;    mitochondrial DNA;    nuclear DNA;    real-time PCR;    quantitative PCR;   

DOI  :  10.1590/S1415-47572009000100003

来源: SciELO

【 摘 要 】

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.

【 授权许可】

CC BY   
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