Real-Time qPCR for Quantification of Circulating mtDNA
Circulating mtDNA is a potential biomarker of cellular mitochondrial dysfunction, which occurs in several human diseases. Changes in mtDNA are usually determined by quantification of mtDNA relative to nuclear DNA (mt/nDNA) using real-time qPCR. methodology and A study has identified that current methods for measuring mt/nDNA need to be improved as they have at least one of the following 3 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 p-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 (Malik et al. 2011). These authors described a qPCR-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, and to accurately quantify mtDNA from human samples.