Characterization of conditions and determination of practical tips for mtDNA level estimation in various human cells

Determination of mtDNA copy number in the cell is crucial to understand many cellular processes. Recently, the number of studies with the use of mitochondrial DNA (mtDNA) content as the determinant of mitochondrial abnormalities increased greatly, and it is still growing, therefore, optimization of technical conditions for this analysis is crucial. Despite using similar laboratory protocols, some results cannot be compared between research centers, thus causing discrepancies in the assessment of mtDNA content. The aim of this work was to test what conditions of biological sample collection and storage affect the estimation of mtDNA level relative to the nuclear DNA (nDNA) in the blood sample and dermal fibroblasts. We found that time and temperature of sample storage as well as type of blood sample (whole blood or leukocytes) influence the estimation of mtDNA/nDNA ratio in the blood. In the case of dermal fibroblasts collected from healthy control and Huntington disease patients, our data indicate that passage number of cells is essential to obtain reliable results.