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2017 | 64 | 4 | 699-704
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Characterization of conditions and determination of practical tips for mtDNA level estimation in various human cells

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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 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 which conditions of biological sample collection and storage affect estimation of mtDNA level relative to the nuclear DNA (nDNA) in the blood samples and dermal fibroblasts. We found that the time and temperature of sample storage, as well as the type of the blood sample (whole blood or leukocytes) influence the estimate 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 the passage number of cells is essential to obtain reliable results.
Physical description
  • Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland
  • Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland
  • Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland
  • Department of Molecular Biology, University of Gdańsk, Gdańsk, Poland
  • Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJ, Staden R, Young IG (1981) Sequence and organization of the human mitochondrial genome. Nature 290: 457-465. doi: 10.1038/290457a0.
  • Andreu AL, Martinez R, Marti R, García-Arumí E (2009) Quantification of mitochondrial DNA copy number: Pre-analytical factors. Mitochondrion 9: 242-246. doi: 10.1016/j.mito.2009.02.006.
  • Battersby BJ, Moyes CD (1998) Influence of acclimation temperature on mitochondrial DNA, RNA, and enzymes in skeletal muscle. Am J Physiol 275: R905-R12.
  • Chabi B, Mousson de Camaret B, Duborjal H, Issartel JP, Stepien G (2003) Quantification of mitochondrial DNA deletion, depletion, and overreplication: application to diagnosis. Clin Chem 49: 1309-1317. doi: 10.1373/49.8.1309.
  • Chen CM, Wu YR, Cheng ML, Liu JL, Lee YM, Lee PW, Soong BW, Chiu DT (2007) Increased oxidative damage and mitochondrial abnormalities in the peripheral blood of Huntington's disease patients. Biochem Biophys Res Commun 359: 335-340. doi: 10.1016/j.bbrc.2007.05.093.
  • Chen S, Li Z, He Y, Zhang F, Li H, Liao Y, Wei Z, Wan G, Xiang X, Hu M, Xia K, Chen X, Tang J (2015) Elevated mitochondrial DNA copy number in peripheral blood cells is associated with childhood autism. BMC Psychiatry 15: 50. doi: 10.1186/s12888-015-0432-y.
  • Chinnery PF, Samuels DC (1999) Relaxed replication of mtDNA: A model with implications for the expression of disease. Am J Hum Genet 64: 1158-1165. doi: 10.1086/302311.
  • Chiu RW, Chan LY, Lam NY, Tsui NB, Ng EK, Rainer TH, Lo YM (2003) Quantitative analysis of circulating mitochondrial DNA in plasma. Clin Chem 49: 719-726. doi: 10.1373/49.5.719.
  • Gonzalez-Hunt CP, Rooney JP, Ryde IT, Anbalagan C, Joglekar R, Meyer JN (2016) PCR-Based analysis of mitochondrial dna copy number, mitochondrial DNA damage, and nuclear DNA damage. Curr Protoc Toxicol 67: 20.11.1-20.11.25. doi: 10.1002/0471140856.tx2011s67.
  • Grady JP, Murphy JL, Blakely EL, Haller RG, Taylor RW, Turnbull DM, Tuppen HA (2014) Accurate measurement of mitochondrial DNA deletion level and copy number differences in human skeletal muscle. PLoS One 9: e114462. doi: 10.1371/journal.pone.0114462.
  • Greaves LC, Reeve AK, Taylor RW, Turnbull DM (2012) Mitochondrial DNA and disease. J Pathol 226: 274-286. doi: 10.1002/path.3028.
  • Holloszy JO, Coyle EF (1984) Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J Appl Physiol 56: 831-838.
  • Huang J, Tan L, Shen R, Zhang L, Zuo H, Wang DW (2016) Decreased Peripheral mitochondrial DNA copy number is associated with the risk of heart failure and long-term outcomes. Medicine (Baltimore) 95: e3323. doi: 10.1097/MD.0000000000003323.
  • Jedrak P, Krygier M, Tońska K, Drozd M, Kaliszewska M, Bartnik E, Sołtan W, Sitek JE, Stanislawska-Sachadyn A, Limon J, Sławek J, Węgrzyn G, Barańska S (2017) Mitochondrial DNA levels in Huntington disease leukocytes and dermal fibroblasts. Metab Brain Dis 32: 1237-1247. doi: 10.1007/s11011-017-0026-0.
  • Kalinowski DP, Illenye S, Van Houten B (1992) Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay. Nucleic Acids Res 20: 3485-3494.
  • Lee H, Song J, Shin C, Park D, Park K, Lee K, Koh CS (1998) Decreased mitochondrial DNA content in peripheral blood precedes the development of non-insulin-dependent diabetes mellitus. Diabetes Res Clin Pract 42: 161-167. doi: 10.1016/S0168-8227(98)00110-7.
  • Lee HC, Yin PH, Lu CY, Chi CW, Wei YH (2000) Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. Biochem J 348: 425-432.
  • Liu CS, Cheng WL, Kuo SJ, Li JY, Soong BW, Wei YH (2008) Depletion of mitochondrial DNA in leukocytes of patients with poly-Q diseases. J Neurol Sci 264: 18-21. doi: 10.1016/j.jns.2007.07.016.
  • Lo YM (2006) Circulating nucleic acids in plasma and serum: an overview. Ann N Y Acad Sci 945: 1-7. doi: 10.1111/j.1749-6632.2001.tb03858.x.
  • Malik AN, Czajka A (2013) Is mitochondrial DNA content a potential biomarker of mitochondrial dysfunction? Mitochondrion 13: 481-492. doi: 10.1016/j.mito.2012.10.011.
  • Malik AN, Shahni R, Iqbal MM (2009) Increased peripheral blood mitochondrial DNA in type 2 diabetic patients with nephropathy. Diabetes Res Clin Pract 86: e22-e24. doi: 10.1016/j.diabres.2009.07.002.
  • Masayesva BG, Mambo E, Taylor RJ, Goloubeva OG, Zhou S, Cohen Y, Minhas K, Koch W, Sciubba J, Alberg AJ, Sidransky D, Califano J (2006) Mitochondrial DNA content increase in response to cigarette smoking. Cancer Epidemiol Biomarkers Prev 15: 19-24. doi: 10.1158/1055-9965.EPI-05-0210.
  • Meissner C, Mohamed SA, Klueter H, Hamann K, von Wurmb N, Oehmichen M (2000) Quantification of mitochondrial DNA in human blood cells using an automated detection system. Forensic Sci Int 113: 109-112. doi: 10.1016/S0379-0738(00)00249-8.
  • Morten K, Ashley N, Wijburg F, Hadzic N, Parr J (2007) Liver mtDNA content increases during development: a comparison of methods and the importance of age-and tissue-specific controls for the diagnosis of mtDNA. Mitochondrion 7: 386-395. doi: 10.1016/j.mito.2007.09.001.
  • Nicholls DG (2002) Mitochondrial bioenergetics, aging, and aging-related disease. Sci Aging Knowl Env 2002: 12. doi: 10.1126/sageke.2002.31.pe12
  • Petersen MH, Budtz-Jørgensen E, Sørensen SA, Nielsen JE, Hjermind LE, Vinther-Jensen T, Nielsen SM, Nørremølle A (2014) Reduction in mitochondrial DNA copy number in peripheral leukocytes after onset of Huntington's disease. Mitochondrion 17: 14-21. doi: 10.1016/j.mito.2014.05.001.
  • Pyle A, Anugrha H, Kurzawa-Akanbi M, Yarnall A, Burn D, Hudson G (2016) Reduced mitochondrial DNA copy number is a biomarker of Parkinson's disease. Neurobiol Aging 38: 216.e7-216.e10. doi: 10.1016/j.neurobiolaging.2015.10.033.
  • Wallace DC (2010) Mitochondrial DNA mutations in disease and aging. Environ Mol Mutagen 51: 440-450. doi: 10.1002/em.20586.
  • Weng SW, Lin TK, Liou CW, Chen SD, Wei YH, Lee HC, Chen IY, Hsieh CJ, Wang PW (2009) Peripheral blood mitochondrial DNA content and dysregulation of glucose metabolism. Diabetes Res Clin Pract 83: 94-99. doi: 10.1016/j.diabres.2008.10.002.
  • Wojtczak L, Zabocki K (2008) Basic mitochondrial physiology in cell viability and death. In Drug-Induced Mitochondrial Dysfunction. Dykens JA Will Y eds, John Wiley & Sons. doi: 10.1002/9780470372531.ch1.
  • Wong J, McLennan SV, Molyneaux L, Min D, Twigg SM, Yue DK (2009) Mitochondrial DNA content in peripheral blood monocytes: relationship with age of diabetes onset and diabetic complications. Diabetologia 52: 1953-1961. doi: 10.1007/s00125-009-1424-6.
  • Zhang H, Cooney DA, Sreenath A, Zhan Q, Agbaria R, Stowe EE, Fornace AJ, Johns DG (1994) Quantitation of mitochondrial DNA in human lymphoblasts by a competitive polymerase chain reaction method: application to the study of inhibitors of mitochondrial DNA content. Mol Pharmacol 46: 1063-1069.
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