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

M E Gahan, Fiona Miller, Sharon R Lewin, Catherine L Cherry, Jennifer F Hoy, A Mijch, F Rosenfeldt, Steven L Wesselingh

Research output: Contribution to journalArticle

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Abstract

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.

OBJECTIVES: 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.

STUDY DESIGN: 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.

Original languageEnglish
Pages (from-to)241-247
Number of pages7
JournalJournal of Clinical Virology
Volume22
Issue number3
Publication statusPublished - Oct 2001
Externally publishedYes

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Subcutaneous Fat
Mitochondrial DNA
Real-Time Polymerase Chain Reaction
Blood Cells
Reverse Transcriptase Inhibitors
Nucleosides
HIV
Biopsy
Toxicity Tests
Ficoll
Peripheral Nervous System Diseases
Centrifugation
Fatigue
Disease Progression
Fats
Cell Line
Muscles
Skin
Mortality
DNA

Cite this

Gahan, M. E., Miller, F., Lewin, S. R., Cherry, C. L., Hoy, J. F., Mijch, A., ... Wesselingh, S. L. (2001). Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction. Journal of Clinical Virology, 22(3), 241-247.
Gahan, M E ; Miller, Fiona ; Lewin, Sharon R ; Cherry, Catherine L ; Hoy, Jennifer F ; Mijch, A ; Rosenfeldt, F ; Wesselingh, Steven L. / Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction. In: Journal of Clinical Virology. 2001 ; Vol. 22, No. 3. pp. 241-247.
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Gahan, ME, Miller, F, Lewin, SR, Cherry, CL, Hoy, JF, Mijch, A, Rosenfeldt, F & Wesselingh, SL 2001, 'Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction', Journal of Clinical Virology, vol. 22, no. 3, pp. 241-247.

Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction. / Gahan, M E; Miller, Fiona; Lewin, Sharon R; Cherry, Catherine L; Hoy, Jennifer F; Mijch, A; Rosenfeldt, F; Wesselingh, Steven L.

In: Journal of Clinical Virology, Vol. 22, No. 3, 10.2001, p. 241-247.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction

AU - Gahan, M E

AU - Miller, Fiona

AU - Lewin, Sharon R

AU - Cherry, Catherine L

AU - Hoy, Jennifer F

AU - Mijch, A

AU - Rosenfeldt, F

AU - Wesselingh, Steven L

PY - 2001/10

Y1 - 2001/10

N2 - 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.OBJECTIVES: 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.STUDY DESIGN: 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.

AB - 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.OBJECTIVES: 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.STUDY DESIGN: 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.

KW - Adipose Tissue

KW - DNA, Mitochondrial

KW - Humans

KW - Leukocytes, Mononuclear

KW - Polymerase Chain Reaction

KW - Reproducibility of Results

KW - Reverse Transcriptase Inhibitors

KW - Taq Polymerase

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

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EP - 247

JO - Clinical and Diagnostic Virology

JF - Clinical and Diagnostic Virology

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