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 attributable to treatment with nucleoside analogue reverse transcriptase inhibitors (NRTIs), attention has now become focused on the toxicities of these forms of treatment. It is believed NRTIs cause a decrease in mitochondrial DNA (mtDNA) synthesis due to their inhibition of DNA polymerase gamma. This hypothesis is supported by in vitro data from muscle biopsies and human lymphoblastic cell lines. The resulting mitochondrial toxicity is thought to manifest itself in a variety of clinical symptoms including fatigue, fat wasting and peripheral neuropathy. A non-invasive test of mitochondrial toxicity is needed to assess toxicity and optimise HIV treatment strategies. Peripheral blood mononuclear cells (PBMC) and subcutaneous fat could be ideal and accessible sources of mtDNA for examining toxicity.


OBJECTIVE
The objectives 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 the utility of subcutaneous fat as a source of mtDNA for quantification.


METHODS
PBMC were isolated from blood by centrifugation over Ficoll-Paque and subcutaneous fat was obtained from two 3 mm punch skin biopsies. Following DNA extraction, the mtDNA copy number in each sample was quantified by real-time polymerase chain reaction (PCR).


RESULTS
The real-time PCR assay was found to generate consistent and reproducible results with replicates of samples undertaken within the same run, and in two or more different runs, having a mean coefficient of variation of 11.3 and 17.2%, respectively. PBMC and subcutaneous fat contained 409+/-148 and 2042+/-391 copies of mtDNA per cell, respectively.


CONCLUSIONS
From the work carried out it can be concluded that firstly, the real-time PCR assay generates consistent and reproducible results, and secondly that mtDNA can be extracted and quantified from PBMC and subcutaneous fat.
Gahan
M E
ME
Infectious Diseases Unit, Alfred Hospital, Department of Medicine Monash University, Prahran, Vic. 3181, Australia.
Miller
F
F
Lewin
S R
SR
Cherry
C L
CL
Hoy
J F
JF
Mijch
A
A
Rosenfeldt
F
F
Wesselingh
S L
SL
D000273:Adipose Tissue / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors; D019914:Taq Polymerase
D004272:DNA, Mitochondrial; D018894:Reverse Transcriptase Inhibitors; D019914:Taq Polymerase