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• W2809075382 abstract "Mitochondrial DNA copies per cell (mtDNA content) can fluctuate with cellular aging, oxidative stress, and mitochondrial dysfunction, and has been investigated in cancer, diabetes, HIV, and metabolic disease. mtDNA content testing in both clinical and basic settings is expected to increase as research uncovers its biological relevance. Herein, we present a novel mtDNA content assay developed on monochrome multiplex real-time quantitative PCR (MMqPCR) principles. This assay offers a greater than twofold improvement on time effectiveness and cost-effectiveness over conventional (monoplex) qPCR, as well as improved reproducibility given the reduced effects of human pipetting errors. The new MMqPCR method was compared with the gold standard monoplex qPCR assay on DNA from a variety of sources, including human whole blood, skeletal muscle, and commercial cell lines. The MMqPCR assay is reproducible (n = 98, r = 0.99, P < 0.0001) and highly correlated to the monoplex qPCR assay (n = 160, r > 0.98, P < 0.0001). Intra-assay and interassay variabilities, as established independently by multiple operators, range between 4.3% and 7.9% and between 2.9% and 9.2%, respectively. This robust assay can quantify >82 pg of template DNA per reaction, with a minimum mtDNA/nuclear DNA ratio of 20, and is especially suitable for studies that require high throughput. Mitochondrial DNA copies per cell (mtDNA content) can fluctuate with cellular aging, oxidative stress, and mitochondrial dysfunction, and has been investigated in cancer, diabetes, HIV, and metabolic disease. mtDNA content testing in both clinical and basic settings is expected to increase as research uncovers its biological relevance. Herein, we present a novel mtDNA content assay developed on monochrome multiplex real-time quantitative PCR (MMqPCR) principles. This assay offers a greater than twofold improvement on time effectiveness and cost-effectiveness over conventional (monoplex) qPCR, as well as improved reproducibility given the reduced effects of human pipetting errors. The new MMqPCR method was compared with the gold standard monoplex qPCR assay on DNA from a variety of sources, including human whole blood, skeletal muscle, and commercial cell lines. The MMqPCR assay is reproducible (n = 98, r = 0.99, P < 0.0001) and highly correlated to the monoplex qPCR assay (n = 160, r > 0.98, P < 0.0001). Intra-assay and interassay variabilities, as established independently by multiple operators, range between 4.3% and 7.9% and between 2.9% and 9.2%, respectively. This robust assay can quantify >82 pg of template DNA per reaction, with a minimum mtDNA/nuclear DNA ratio of 20, and is especially suitable for studies that require high throughput. CME Accreditation Statement: This activity (“JMD 2018 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2018 CME Program in Molecular Diagnostics”) for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“JMD 2018 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2018 CME Program in Molecular Diagnostics”) for a maximum of 18.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Mitochondria contain multiple copies of circular mitochondrial DNA (mtDNA) that code for some of the proteins necessary for oxidative phosphorylation, as well as 22 tRNAs and two rRNAs.1Anderson S. Bankier A.T. Barrell B.G. de Bruijn M.H. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Sequence and organization of the human mitochondrial genome.Nature. 1981; 290: 457-465Crossref PubMed Scopus (7631) Google Scholar mtDNA replicates independently of both cellular division and mitochondrial proliferation, although the number of mtDNA copies per cell is typically maintained within a homeostatic range.2Fukuhara H. Relative proportions of mitochondrial and nuclear DNA in yeast under various conditions of growth.Eur J Biochem. 1969; 11: 135-139Crossref PubMed Scopus (59) Google Scholar, 3Moraes C.T. Kenyon L. Hao H. Mechanisms of human mitochondrial DNA maintenance: the determining role of primary sequence and length over function.Mol Biol Cell. 1999; 10: 3345-3356Crossref PubMed Scopus (71) Google Scholar The copy number of mtDNA per cell can be modulated on the basis of the cell's energetic needs as well as the presence of the nuclear-encoded mitochondrial transcription factor A, which acts on the promoter regions of mtDNA to regulate replication and transcription.4Fisher R.P. Clayton D.A. A transcription factor required for promoter recognition by human mitochondrial RNA polymerase: accurate initiation at the heavy- and light-strand promoters dissected and reconstituted in vitro.J Biol Chem. 1985; 260: 11330-11338PubMed Google Scholar, 5Virbasius J.V. Scarpulla R.C. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis.Proc Natl Acad Sci U S A. 1994; 91: 1309-1313Crossref PubMed Scopus (613) Google Scholar, 6Lee H.-C. Wei Y.-H. Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress.Int J Biochem Cell Biol. 2005; 37: 822-834Crossref PubMed Scopus (481) Google Scholar Altered levels of mtDNA copies per cell (mtDNA content) in various tissues have been associated with aging7Mengel-From J. Thinggaard M. Dalgard C. Kyvik K.O. Christensen K. Christiansen L. Mitochondrial DNA copy number in peripheral blood cells declines with age and is associated with general health among elderly.Hum Genet. 2014; 133: 1149-1159Crossref PubMed Scopus (210) Google Scholar, 8Barrientos A. Casademont J. Cardellach F. Estivill X. Urbano-Marquez A. Nunes V. Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging.Mol Brain Res. 1997; 52: 284-289Crossref PubMed Scopus (128) Google Scholar and may be a physiological response to oxidative stress.9Lee H.C. Yin P.H. Lu C.Y. Chi C.W. Wei Y.H. Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells.Biochem J. 2000; 348 Pt 2: 425-432Crossref PubMed Scopus (449) Google Scholar, 10Lee H.C. Lu C.Y. Fahn H.J. Wei Y.H. Aging- and smoking-associated alteration in the relative content of mitochondrial DNA in human lung.FEBS Lett. 1998; 441: 292-296Crossref PubMed Scopus (129) Google Scholar mtDNA content has been examined in the context of numerous diseases and conditions. For example, measurable changes in mtDNA content have been reported in various forms of cancer,11Wang Y. Liu V.W. Xue W.C. Cheung A.N. Ngan H.Y. Association of decreased mitochondrial DNA content with ovarian cancer progression.Br J Cancer. 2006; 95: 1087-1091Crossref PubMed Scopus (118) Google Scholar, 12Meierhofer D. Decrease of mitochondrial DNA content and energy metabolism in renal cell carcinoma.Carcinogenesis. 2004; 25: 1005-1010Crossref PubMed Scopus (132) Google Scholar, 13He X. Qu F. Zhou F. Zhou X. Chen Y. Guo X. High leukocyte mtDNA content contributes to poor prognosis through ROS-mediated immunosuppression in hepatocellular carcinoma patients.Oncotarget. 2014; 7: 22834-22845Google Scholar in diabetes,14Lee H.K. Song J.H. Shin C.S. Park D.J. Park K.S. Lee K.U. Koh C.S. Decreased mitochondrial DNA content in peripheral blood precedes the development of non-insulin-dependent diabetes mellitus.Diabetes Res Clin Pract. 1998; 42: 161-167Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 15Song J. Oh J.Y. Sung Y.A. Pak Y.K. Park K.S. Lee H.K. Peripheral blood mitochondrial DNA content is related to insulin sensitivity in offspring of type 2 diabetic patients.Diabetes Care. 2001; 24: 865-869Crossref PubMed Scopus (118) Google Scholar, 16Malik A.N. Parsade C.K. Ajaz S. Crosby-Nwaobi R. Gnudi L. Czajka A. Sivaprasad S. Altered circulating mitochondrial DNA and increased inflammation in patients with diabetic retinopathy.Diabetes Res Clin Pract. 2015; 110: 257-265Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar and in HIV-infected individuals either untreated or treated with certain antiretroviral therapy regimens.17Côté H.C. Brumme Z.L. Craib K.J. Alexander C.S. Wynhoven B. Ting L. Wong H. Harris M. Harrigan P.R. O'Shaughnessy M.V. Montaner J.S. Changes in mitochondrial DNA as a marker of nucleoside toxicity in HIV-infected patients.N Engl J Med. 2002; 346: 811-820Crossref PubMed Scopus (516) Google Scholar, 18Miura T. Goto M. Hosoya N. Odawara T. Kitamura Y. Nakamura T. Iwamoto A. Depletion of mitochondrial DNA in HIV-1-infected patients and its amelioration by antiretroviral therapy.J Med Virol. 2003; 70: 497-505Crossref PubMed Scopus (90) Google Scholar, 19Miró O. López S. Martínez E. Pedrol E. Milinkovic A. Deig E. Garrabou G. Casademont J. Gatell J.M. Cardellach F. Mitochondrial effects of HIV infection on the peripheral blood mononuclear cells of HIV-infected patients who were never treated with antiretrovirals.Clin Infect Dis. 2004; 39: 710-716Crossref PubMed Scopus (127) Google Scholar Some studies demonstrate the depletion of mtDNA content by HIV infection longitudinally.18Miura T. Goto M. Hosoya N. Odawara T. Kitamura Y. Nakamura T. Iwamoto A. Depletion of mitochondrial DNA in HIV-1-infected patients and its amelioration by antiretroviral therapy.J Med Virol. 2003; 70: 497-505Crossref PubMed Scopus (90) Google Scholar, 20Casula M. Bosboom-Dobbelaer I. Smolders K. Otto S. Bakker M. de Baar M.P. Reiss P. de Ronde A. Infection with HIV-1 induces a decrease in mtDNA.J Infect Dis. 2005; 191: 1468-1471Crossref PubMed Scopus (54) Google Scholar mtDNA quantification is also of particular interest as a potential marker for mitochondrial dysfunction in critical care21Cote H.C. Day A.G. Heyland D.K. Longitudinal increases in mitochondrial DNA levels in blood cells are associated with survival in critically ill patients.Crit Care. 2007; 11: R88Crossref PubMed Scopus (17) Google Scholar as well as metabolic comorbidities, such as hyperlactatemia,22Miro O. Lopez S. Martinez E. Rodriguez-Santiago B. Blanco J.L. Milinkovic A. Miro J.M. Nunes V. Casademont J. Gatell J.M. Cardellach F. Reversible mitochondrial respiratory chain impairment during symptomatic hyperlactatemia associated with antiretroviral therapy.AIDS Res Hum Retroviruses. 2003; 19: 1027-1032Crossref PubMed Scopus (15) Google Scholar myopathy and cardiomyopathy,23Tritschler H. Andreetta F. Moraes C. Bonilla E. Arnaudo E. Danon M. Glass S. Zelaya B. Vamos E. Telerman-Toppet N. Shanske S. Kadenbach B. DiMauro S. Schon E. Mitochondrial myopathy of childhood associated with depletion of mitochondrial DNA.Neurology. 1992; 42: 209-217Crossref PubMed Google Scholar, 24Arnaudo E. Shanske S. DiMauro S. Schon E.A. Moraes C.T. Schon E.A. Dalakas M. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy.Lancet. 1991; 337: 508-510Abstract PubMed Scopus (470) Google Scholar, 25Pisano A. Cerbelli B. Perli E. Pelullo M. Bargelli V. Preziuso C. Mancini M. He L. Bates M.G. Lucena J.R. Della Monica P.L. Familiari G. Petrozza V. Nediani C. Taylor R.W. D’Amati G. Giordano C. Impaired mitochondrial biogenesis is a common feature to myocardial hypertrophy and end-stage ischemic heart failure.Cardiovasc Pathol. 2016; 25: 103-112Crossref PubMed Scopus (65) Google Scholar peripheral and optic neuropathies,26Dalakas M.C. Semino-Mora C. Leon-Monzon M. Mitochondrial alterations with mitochondrial DNA depletion in the nerves of AIDS patients with peripheral neuropathy induced by 2′3′-dideoxycytidine (ddC).Lab Invest. 2001; 81: 1537-1544Crossref PubMed Scopus (183) Google Scholar, 27Yen M. Chen C. Wang A. Wei Y. Increase of mitochondrial DNA in blood cells of patients with Leber’s hereditary optic neuropathy with 11778 mutation.Br J Ophthalmol. 2002; 86: 1027-1030Crossref PubMed Scopus (37) Google Scholar and lipodystrophy.28Shikuma C.M. Hu N. Milne C. Yost F. Waslien C. Shimizu S. Shiramizu B. Mitochondrial DNA decrease in subcutaneous adipose tissue of HIV-infected individuals with peripheral lipoatrophy.AIDS. 2001; 15: 1801-1809Crossref PubMed Scopus (191) Google Scholar Furthermore, reduced mtDNA content is a hallmark of mitochondrial DNA depletion syndrome, a heterogeneous set of genetic disorders that may clinically present as hepatic failure, encephalopathy, and myopathy.29Spinazzola A. Zeviani M. Disorders of nuclear-mitochondrial intergenomic communication.Biosci Rep. 2007; 27: 39-51Crossref PubMed Scopus (60) Google Scholar, 30Dimmock D. Tang L.Y. Schmitt E.S. Wong L.J.C. Quantitative evaluation of the mitochondrial DNA depletion syndrome.Clin Chem. 2010; 56: 1119-1127Crossref PubMed Scopus (106) Google Scholar To date, the usefulness of mtDNA content as a diagnostic and/or molecular screening tool remains a matter of debate because of the current paucity of mechanistic evidence in various disease states, unknown sensitivity and specificity, and a scarcity of prospective cohort studies demonstrating causality.31Yu M. Generation, function and diagnostic value of mitochondrial DNA copy number alterations in human cancers.Life Sci. 2011; 89: 65-71Crossref PubMed Scopus (159) Google Scholar, 32Wen S.L. Zhang F. Feng S. Decreased copy number of mitochondrial DNA: a potential diagnostic criterion for gastric cancer.Oncol Lett. 2013; 6: 1098-1102Crossref PubMed Scopus (33) Google Scholar, 33Fernandes J. Michel V. Camorlinga-Ponce M. Gomez A. Maldonado C. De Reuse H. Torres J. Touati E. Circulating mitochondrial DNA level, a noninvasive biomarker for the early detection of gastric cancer.Cancer Epidemiol Biomarkers Prev. 2014; 23: 2430-2438Crossref PubMed Scopus (32) Google Scholar Nevertheless, it is likely that the use of mtDNA content measurements in both clinical and research settings will become more prevalent as data are generated and their biological relevance is more clearly established. Real-time quantitative PCR (qPCR) is the preferred method of measuring mtDNA content because of its applicability in both fresh and archived tissues, its high-throughput nature, and extensive use history for more than a decade. In 2009, Cawthon34Cawthon R.M. Telomere length measurement by a novel monochrome multiplex quantitative PCR method.Nucleic Acids Res. 2009; 37: 1-7Crossref PubMed Scopus (954) Google Scholar described a monochrome multiplex qPCR (MMqPCR) technique for the measurement of telomere length, exploiting copy number differences and melting temperatures of single-copy and telomeric DNA amplicons. Our group has previously described optimization conditions for this technique on the LightCycler 480 platform (Roche Molecular Systems, Pleasanton, CA) with SYBR Green intercalating fluorescent dye.35Hsieh A.Y.Y. Saberi S. Ajaykumar A. Hukezalie K. Gadawski I. Sattha B. Cote H.C.F. Optimization of a relative telomere length assay by monochromatic multiplex real-time quantitative PCR on the LightCycler 480: sources of variability and quality control considerations.J Mol Diagn. 2016; 18: 425-437Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Herein, we implement the MMqPCR method for the measurement of mtDNA content on the basis of the same principles. We present quality control considerations and quantify assay variance, and we juxtapose the performance of this assay relative to the gold standard monoplex qPCR. This study was approved by the University of British Columbia Research Ethics Board. All study participants and volunteer blood donors provided written informed consent. Human tissues (n = 130) and cultured human cell specimens (n = 30) were used in this study. Our sample of convenience consisted of human DNA from the following tissues: whole blood (WB; n = 46), mouth swab (n = 30), as well as biopsy samples of skeletal muscle (n = 35), subcutaneous fat (n = 4), heart (n = 4), liver (n = 4), lung (n = 4), and kidney (n = 3). These were randomly selected from a DNA biobank of specimens from previously published studies.36Côté H. Brumme Z. Chan J. Guillemi S. Montaner J. Harrigan P. HIV therapy, hepatitis C virus infection, antibiotics and obesity, a mitochondria killer mix?.AIDS. 2006; 20: 1343-1345Crossref PubMed Scopus (2) Google Scholar, 37Stringer H.A.J. Sohi G.K. Maguire J.A. Côté H.C.F. Decreased skeletal muscle mitochondrial DNA in patients with statin-induced myopathy.J Neurol Sci. 2013; 325: 142-147Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 38Schick B.A. Laaksonen R. Frohlich J.J. Päivä H. Lehtimäki T. Humphries K.H. Côté H.C.F. Decreased skeletal muscle mitochondrial DNA in patients treated with high-dose simvastatin.Clin Pharmacol Ther. 2007; 81: 650-653Crossref PubMed Scopus (87) Google Scholar In addition, cultured human cells were used: JEG-3 (ATCC-HTB-36) and BeWo (ATCC-CCL-98) placental cells, CCRF-CEM (ATCC-CRM-CCL-119) T-lymphoblast cells, and HEK-293 (ATCC-CRL-1573) embryonic kidney cells. As part of other studies, cultured cells had been exposed to HIV antiretrovirals, some of which affect mtDNA content.17Côté H.C. Brumme Z.L. Craib K.J. Alexander C.S. Wynhoven B. Ting L. Wong H. Harris M. Harrigan P.R. O'Shaughnessy M.V. Montaner J.S. Changes in mitochondrial DNA as a marker of nucleoside toxicity in HIV-infected patients.N Engl J Med. 2002; 346: 811-820Crossref PubMed Scopus (516) Google Scholar Total genomic DNA from WB (0.1 mL) and cultured cells (approximately 1 × 106 cells) was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) on the QIAcube, according to the manufacturer's blood and body fluid protocol, with modifications as previously described.39Zanet D.L. Saberi S. Oliveira L. Sattha B. Gadawski I. Côté H.C. Blood and dried blood spot telomere length measurement by qPCR: assay considerations.PLoS One. 2013; 8: e57787Crossref PubMed Scopus (54) Google Scholar Total DNA from muscle, fat, heart, liver, lung, and kidney tissues was collected and extracted as described in previous studies.36Côté H. Brumme Z. Chan J. Guillemi S. Montaner J. Harrigan P. HIV therapy, hepatitis C virus infection, antibiotics and obesity, a mitochondria killer mix?.AIDS. 2006; 20: 1343-1345Crossref PubMed Scopus (2) Google Scholar, 37Stringer H.A.J. Sohi G.K. Maguire J.A. Côté H.C.F. Decreased skeletal muscle mitochondrial DNA in patients with statin-induced myopathy.J Neurol Sci. 2013; 325: 142-147Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 38Schick B.A. Laaksonen R. Frohlich J.J. Päivä H. Lehtimäki T. Humphries K.H. Côté H.C.F. Decreased skeletal muscle mitochondrial DNA in patients treated with high-dose simvastatin.Clin Pharmacol Ther. 2007; 81: 650-653Crossref PubMed Scopus (87) Google Scholar mtDNA content was defined as the ratio of the copy number of mitochondrial genomes normalized to the copy number of a single-copy nuclear gene. Fragments of the mitochondrial displacement loop (D-loop) and albumin (ALB) were used as mtDNA and nuclear DNA (nDNA) sequences, respectively. Both sequences were amplified in a single 10-μL reaction containing 1× LightCycler SYBR Green I Master (product number 04707516001; Roche Molecular Systems), 0.9 μmol/L of each of the following four primers (AlbuF, AlbdR, D-loop_MPLX_F, and D-loop_MPLX_R), 1.2 mmol/L EDTA, and 2 μL of genomic DNA template (approximately 0.3 to 35 ng/μL). Primers were high-performance liquid chromatography purified (Integrated DNA Technologies, Coralville, IA), and their sequences are presented in Table 1. Noncomplementary tags were added to the 5′ end of the primer sequences to modify amplicon melting temperatures. Degenerate bases in D-loop primers were incorporated to accommodate common mtDNA sequence variants, as reported in Mitomap (http://www.mitomap.org, last accessed October 4, 2017), an integrated database of human mtDNA sequence variants.40Kogelnik A. MITOMAP: a human mitochondrial genome database.Nucleic Acids Res. 1996; 24: 177-179Crossref PubMed Scopus (120) Google Scholar The thermal cycling program started with a 95°C enzyme activation (hot-start) incubation for 15 minutes and was followed by 40 cycles of 94°C for 15 seconds, 62°C for 10 seconds (2.2°C/second), 74°C for 15 seconds, 84°C for 10 seconds, and 88°C for 15 seconds, with two signal acquisitions at the end of the 74°C and 88°C stages. Temperature ramping rates were all 4.4°C/second unless specified.Table 1Primer SequencesAssay typeTargetAccession no.∗https://www.ncbi.nlm.nih.gov.Amplicon size, bpForward primerForward primer nucleotide sequenceReverse primerReverse primer nucleotide sequenceMonoplex qPCRMT-CO1NC_012920.1197CCOI1F5′-TTCGCCGACCGTTGACTATT-3′CCOI2R5′-AAGATTATTACAAATGCATGGGC-3′Monoplex qPCRPOLG2NM_007215.3186ASPG3F5′-GAGCTGTTGACGGAAAGGAG-3′ASPG4R5′-CAGAAGAGAATCCCGGCTAAG-3′Monoplex qPCRD-loopNC_012920.1150MT325F5′-CACAGCACTTAAACACATCTCTGC-3′MT474R5′-AGTATGGGAGTGRGAGGGRAAAA-3′Monoplex qPCRALBNM_000477.544AlbuFM5′-AAATGCTGCACAGAATCCTTG-3′AlbdRM5′-GAAAAGCATGGTCGCCTGTT-3′MMqPCRALBNM_000477.598AlbuF5′-CGGCGGCGGGCGGCGCGGGCTGGGCGGAAATGCTGCACAGAATCCTTG-3′AlbdR5′-GCCCGGCCCGCCGCGCCCGTCCCGCCGGAAAAGCATGGTCGCCTGTT-3′MMqPCRD-loopNC_012920.1173D-loop_MPLX_F5′-ACGCTCGACACACAGCACTTAAACACATCTCTGC-3′D-loop_MPLX_R5′-GCTCAGGTCATACAGTATGGGAGTGRGAGGGRAAAA-3′Boldfaced nucleotides represent noncomplementary GC clamps, and underlined nucleotides represent noncomplementary bases added to increase the melting temperature of the amplicon. Degenerate (50% A and 50% G) bases are represented by R.∗ https://www.ncbi.nlm.nih.gov. Open table in a new tab Boldfaced nucleotides represent noncomplementary GC clamps, and underlined nucleotides represent noncomplementary bases added to increase the melting temperature of the amplicon. Degenerate (50% A and 50% G) bases are represented by R. A standard curve was included on each plate (r > 0.999), consisting of a mixture of two pCR2.1-TOPO plasmids, each containing one of the amplicons. The two plasmids were mixed at a 50:1 ratio of D-loop/Alb, serially diluted 1:5 to obtain a range of 3250 to 1.27 × 109 copies of D-loop and 65 to 2.5 × 107 copies of ALB. A total of 40 DNA specimens were assayed in duplicate in each run. Data were acquired on the LightCycler 480 software version 1.5.1.62 SP2. The signal acquired at 88°C is reflective of only ALB amplicon copy number, given that the D-loop amplicon dissociates at a lower temperature. Although the 74°C signal technically represents the sum of both D-loop and ALB amplicons, the quantity of ALB is negligible at cycle threshold (CT) given the usually much higher amount of D-loop. Therefore, the 74°C CT can be used to calculate D-loop copy number, and the 88°C CT can be used to calculate ALB copy number. Data from an MMqPCR run were exported from the LightCycler 480 software as one text file and imported into Microsoft Excel (Microsoft Corp., Redmond, WA) to delineate the D-loop (74°C) and ALB (88°C) signals with the program and segment markers specified by the LightCycler 480 software. Using the thermal cycling program described herein, D-loop data are recorded in program 2, segment 3, and ALB data are recorded in program 2, segment 5. Once separated, data for each amplicon were individually exported as text files and converted into grid format using LC480 Conversion version 2.0. Baseline corrections and CT determination were done on the LinRegPCR software version 2012.1, as previously described.41Ruijter J.M. Ramakers C. Hoogaars W.M. Karlen Y. Bakker O. Van Den Hoff M.J. Moorman A.F. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.Nucleic Acids Res. 2009; 37: e45Crossref PubMed Scopus (2165) Google Scholar Both programs are freely available from the Heart Failure Research Center (Amsterdam, the Netherlands; http://www.hartfaalcentrum.nl/index.php?main=files&sub=0, last accessed September 25, 2017). More details regarding data analysis workflow can be found in a previous publication by our group.35Hsieh A.Y.Y. Saberi S. Ajaykumar A. Hukezalie K. Gadawski I. Sattha B. Cote H.C.F. Optimization of a relative telomere length assay by monochromatic multiplex real-time quantitative PCR on the LightCycler 480: sources of variability and quality control considerations.J Mol Diagn. 2016; 18: 425-437Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Monoplex qPCR was based on a previously described fluorescein probe method17Côté H.C. Brumme Z.L. Craib K.J. Alexander C.S. Wynhoven B. Ting L. Wong H. Harris M. Harrigan P.R. O'Shaughnessy M.V. Montaner J.S. Changes in mitochondrial DNA as a marker of nucleoside toxicity in HIV-infected patients.N Engl J Med. 2002; 346: 811-820Crossref PubMed Scopus (516) Google Scholar adapted to the SYBR intercalating dye. DNA samples were assayed twice by monoplex qPCR, amplifying two different sets of mtDNA and nDNA amplicons each time, each gene in a separate qPCR. In the first set, primers complementary to the cytochrome c oxidase subunit 1 (MT-CO1: CCOI1F and CCOI2R) and polymerase (DNA-directed), γ2, accessory subunit (POLG2: ASPG3F and ASPG4R) were used as the mitochondrial and single-copy nuclear genes, respectively. In the second set, the same D-loop (MT325F and MT474R) and ALB (AlbuFM and AlbdRM) sequences as above were amplified, although the D-loop and ALB monoplex primers did not contain any noncomplementary tags (Table 1). Quantification of both genes within a set took place in different wells but on the same plate. Each 10-μL reaction consisted of 1× LightCycler SYBR Green I Master, 1 μmol/L each of forward and reverse primers, and 2 μL of genomic DNA template (approximately 0.3 to 35 ng/μL). The thermal cycling program started with 95°C enzyme activation (hot-start) incubation for 10 minutes. This was followed by 45 cycles of 95°C for 5 seconds, 60°C for 10 seconds (2.2°C/second), and 74°C for 5 seconds, with signal acquisition at the end of the 60°C stage and temperature ramping at 4.4°C/second unless indicated. Data were acquired on the LightCycler 480 software version 1.5.1.62 SP2. A standard curve for each gene was included on each plate. The standard curves for the MT-CO1/POLG2 set consisted of 1:10 serial dilutions of a cloned plasmid (pCR2.1-TOPO; Invitrogen, Carlsbad, CA) containing a single copy of both MT-COI and POLG2 amplicons, with a linear range of 4.3 × 100 to 4.3 × 106 copies (r > 0.999). For the D-loop/Alb set, the same standard curve as for MMqPCR was used for convenience. A total of 17 DNA specimens were assayed in duplicate per run. Data from each gene were independently exported from the LightCycler 480 software as text files and processed with LC480 Conversion and LinRegPCR, as described in the MMqPCR assay above. The data do not need to be sorted by acquisition because each amplicon was measured in separate PCRs. In-house quality control (QC) practices have been established for this assay. These involve a run-specific QC followed by a specimen-specific QC. To ensure the quality of any given run, two internal controls (ICs) and a negative control were included on each plate. One IC was composed of pooled WB DNA extracts from 12 volunteers, and the other was DNA extracted from SK-BR-3 (ATCC-HTB-30) mammary gland epithelial cells. The negative control consisted of DNA elution buffer (Buffer AE; Qiagen) in place of template DNA. For an MMqPCR run to pass QC, it must not violate more than one of the following criteria: i) IC measurements in each run must fall within 2 SDs of IC measurements from previous runs performed under the same conditions; ii) PCR efficiencies of each gene (as determined by the standard curve) must each lie between 90% and 100% (1.80- to 2.00-fold amplification per cycle); iii) differences in PCR efficiencies between the two genes must be <2.5%; iv) the mean of the difference between duplicate mtDNA content measurements must be <10%; and v) signal in the negative control must be absent or negligible (>3CT below the last standard). Specimen-specific QC was based on variation between specimen duplicates. For MMqPCR, data for individual extracts are accepted if mtDNA content measurements (D-loop/Alb ratio) between duplicates varies by <15%. In monoplex qPCR, data for individual extracts are accepted if both individual genes and their ratio (mtDNA content) measurements between duplicates vary by <20%. For both methods, DNA extracts that do not meet QC criteria are repeated in duplicate. If the repeated measurement sti" @default.
• W2809075382 created "2018-06-29" @default.
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• W2809075382 date "2018-09-01" @default.
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• W2809075382 title "A Monochrome Multiplex Real-Time Quantitative PCR Assay for the Measurement of Mitochondrial DNA Content" @default.
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