Quantitative analysis of human mitochondrial DNA using a real-time PCR assay

Abstract

Objectives: Known for their ability to inhibit the human DNA polymerase-gamma, nucleoside analogues induce toxic effects on mitochondria ranging from increased serum lactate levels to fatal lactic acidosis. DNA polymerase-gamma ensures the mitochondrial DNA (mtDNA) replication and, thus, its inhibition leads to the decrease of the mtDNA. We describe a real-time PCR assay for mtDNA quantification associating DNA extraction procedures applied on peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues and to study the antiretroviral effect on mitochondria.

Methods: Total DNA was extracted from PBMCs and subcutaneous adipose tissues. Nuclear and mitochondrial genes were amplified to determine the number of copies of mtDNA per cell using a cyt-b recombinant plasmid as standard control. We analysed eight HIV-infected asymptomatic patients never treated, four patients who had been treated for 6 months with highly active antiretroviral therapy (HAART) and six non-infected donors.

Results: The mtDNA quantification gave rise to reproducible results as the mean coefficients of variation were 1.09% for replicates of samples undertaken 10 times within the same run, and 5.78% and 3.7% for replicates tested in five different runs at 1:100 and 1:1000 dilutions, respectively. Median levels of mtDNA in PBMCs of healthy donors, naive and treated HIV-infected patients were 2.94, 2.78 and 1.93 log HIV-1 RNA copies/mL, respectively. Whereas DNA from PBMCs was shown to be devoid of inhibitors, subcutaneous adipose tissues needed an extra treatment as they were found to be highly inhibited.

Conclusions: The method generated consistent and reproducible results and was successfully applied to DNAs extracted from PBMCs and subcutaneous adipose tissues with adapted extraction. The mtDNA changes in PBMCs were found to be fast as they fall off after 6 months' therapy, decreasing from 2.78 to 1.93 log copies/mL.