Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction

Background: With decreased rates of HIV mortality and disease progression a ttributable to treatment with nucleoside analogue reverse transcriptase inh ibitors (NRTIs), attention has now become focused on the toxicities of thes e forms of treatment. It is believed NRTIs cause a decrease in mitochondria l DNA (mtDNA) synthesis due to their inhibition of DNA polymerase gamma. Th is hypothesis is supported by in vitro data from muscle biopsies and human lymphoblastic cell lines. The resulting mitochondrial toxicity is thought t o manifest itself in a variety of clinical symptoms including fatigue, fat wasting and peripheral neuropathy. A non-invasive test of mitochondrial tox icity is needed to assess toxicity and optimise HIV treatment strategies. P eripheral blood mononuclear cells (PBMC) and subcutaneous fat could be idea l and accessible sources of mtDNA for examining toxicity. Objectives: The o bjectives of this study were (a) to develop an assay to quantify the mtDNA copy number of PBMC and obtain reproducible results and (b) to establish th e utility of subcutaneous fat as a source of mtDNA for quantification. Stud y Design: PBMC were isolated from blood by centrifugation over Ficoll-Paque ((R)) and subcutaneous fat was obtained from two 3 mm, punch skin biopsies. Following DNA extraction, the mtDNA copy number in each sample was quantif ied by real-time polymerase chain reaction (PCR). Results: The real-time PC R assay was found to generate consistent and reproducible results with repl icates of samples undertaken within the same run, and in two or more differ ent runs, having a mean coefficient of variation of 11.3 and 17.2%, respect ively. PBMC and subcutaneous fat contained 409 +/- 148 and 2042 +/- 391 cop ies of mtDNA per cell, respectively. Conclusions: From the work carried out it can be concluded that firstly, the real-time PCR assay generates consis tent and reproducible results, and secondly that mtDNA can be extracted and quantified from PBMC and subcutaneous fat. (C) 2001 Elsevier Science B.V. All rights reserved.