title: Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.
abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.
authors.affiliation: King's College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine, UK. afshan.malik@kcl.ac.uk
authors.forename: Afshan N
authors.initials: AN
authors.lastname: Malik
authors.forename: Rojeen
authors.initials: R
authors.lastname: Shahni
authors.forename: Ana
authors.initials: A
authors.lastname: Rodriguez-de-Ledesma
authors.forename: Abas
authors.initials: A
authors.lastname: Laftah
authors.forename: Phil
authors.initials: P
authors.lastname: Cunningham
mesh_terms: D002460:Cell Line; D002477:Cells / Q000737:chemistry; D004272:DNA, Mitochondrial / Q000032:analysis*; D005819:Genetic Markers; D015894:Genome, Human; D054629:Genome, Mitochondrial / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011544:Pseudogenes
chemical_list: D004272:DNA, Mitochondrial; D005819:Genetic Markers