Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR
Open Access
- 1 January 2009
- journal article
- Published by FapUNIFESP (SciELO) in Genetics and Molecular Biology
- Vol. 32 (1), 20-24
- https://doi.org/10.1590/S1415-47572009000100003
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 efficiencyKeywords
This publication has 25 references indexed in Scilit:
- Maternal SmokingObstetrics & Gynecology, 2007
- Quantitative analysis of intact fetal cells in maternal plasma by real-time PCREuropean Journal of Obstetrics & Gynecology and Reproductive Biology, 2007
- Elevated level of cell-free plasma DNA is associated with breast cancerArchiv für Gynäkologie, 2007
- Regulation of mitochondrial DNA content and cancerMitochondrion, 2006
- Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identificationAnalytical Biochemistry, 2005
- Circulating Nucleic Acids in Plasma or Serum (CNAPS) as Prognostic and Predictive Markers in Patients with Solid NeoplasiasDisease Markers, 2005
- Detection of Mitochondrial DNA Alterations in Plasma of Malignant Melanoma PatientsAnnals of the New York Academy of Sciences, 2004
- Oxidative Stress-related Alteration of the Copy Number of Mitochondrial DNA in Human LeukocytesFree Radical Research, 2003
- Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsiaAmerican Journal of Obstetrics and Gynecology, 2001
- Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutationsProceedings of the National Academy of Sciences of the United States of America, 1997