DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

Influence of cell distribution and diabetes status on the association between mitochondrial DNA copy number and aging phenotypes in the InCHIANTI study

Mitochondrial DNA copy number (mtDNA‐CN) estimated in whole blood is a novel marker of mitochondrial mass and function that can be used in large population‐based studies. Analyses that attempt to relate mtDNA‐CN to specific aging phenotypes may be confounded by differences in the distribution of blood cell types across samples. Also, low or high mtDNA‐CN may have a different meaning given the presence of diseases associated with mitochondrial damage. We evaluated the impact of blood cell type distribution and diabetes status on the association between mtDNA‐CN and aging phenotypes, namely chronologic age, interleukin‐6, hemoglobin, and all‐cause mortality, among 672 participants of the InCHIANTI study. After accounting for white blood cell count, platelet count, and white blood cell proportions in multivariate models, associations of mtDNA‐CN with age and interleukin‐6 were no longer statistically significant. Evaluation of a statistical interaction by diabetes status suggested heterogeneity of effects in the analysis of mortality (P < 0.01). The magnitude and direction of associations between mtDNA‐CN estimated from blood samples and aging phenotypes are influenced by the sample cell type distribution and disease status. Therefore, accounting for these factors may aid understanding of the relevance of mitochondrial DNA copy number to health and aging.     

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.     

qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.     

乳头状甲状腺癌中线粒体DNA突变的研究

该文探究了线粒体DNA(mtDNA)突变与甲状腺癌的发生发展的相关性,评估了mtDNA拷贝数对甲状腺癌的诊断价值。根据对结节性甲状腺肿、滤泡状甲状腺腺瘤和乳头状甲状腺癌3组病人的mtDNA全基因测序和单倍型分型结果,统计3组病人mtDNA突变率及单倍型的差异,分析乳头状甲状腺癌病人的mtDNA突变率与临床资料的联系,最后通过荧光定量PCR检测3组病人的组织和血液样本中mtDNA的拷贝数。结果显示,乳头状甲状腺癌患者mtDNA的复合体I亚基编码区和tRNA编码区的突变率明显高于结节性甲状腺肿,在乳头状甲状腺癌患者中线粒体单体型M相对于单体型N有更低的淋巴结转移率,荧光定量PCR结果显示,甲状腺腺瘤和甲状腺癌组织中的mtDNA拷贝数明显高于结节性甲状腺肿,而在血液标本中,两者的mtDNA拷贝数均低于结节性甲状腺肿。这些结果表明,mtDNA拷贝数的变化和复合体I亚基编码区的突变可能作为甲状腺癌诊断的生物指标,而线粒体单体型N可能可以作为乳头状甲状腺癌恶性变化的预警指标。     

Quality assessment of human mitochondrial DNA quantification: MITONAUTS, an international multicentre survey

Mitochondrial DNA quantification by qPCR is used in the context of many diseases and toxicity studies but comparison of results between laboratories is challenging. Through two multigroup distributions of DNA samples from human cell lines, the MITONAUTS group anonymously compared mtDNA/nDNA quantification across nine laboratories involved in HIV research worldwide. Eight of the nine sites showed significant correlation between them (mean raw data R(2)=0.664; log(10)-transformed data R(2)=0.844). Although mtDNA/nDNA values were well correlated between sites, the inter-site variability on the absolute measurements remained high with a mean (range) coefficient of variation of 71 (37-212) %. Some variability appeared cell line-specific, probably due to chromosomal alterations or pseudogenes affecting the quantification of certain genes, while within cell line variability was likely due to differences in calibration of the standard curves. The use of two mtDNA and two single copy nDNA genes with highly specific primers to quantify each genome would help address copy number variants. Our results indicate that sample shipment must be done frozen and that absolute mtDNA/nDNA ratio values cannot readily be compared between laboratories, especially if assessing cultured cell mtDNA content. However, within laboratory and relative mtDNA/nDNA comparisons between laboratories should be reliable.     

基于数字PCR的不同品种鸭组织中线粒体与核DNA拷贝数差异研究

肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。     

Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.     

荧光定量PCR精确检测人胚胎干细胞线粒体DNA拷贝数方法的建立

建立一种精确定量人胚胎干细胞线粒体DNA拷贝数的方法。构建包含线粒体DNANDl和核单拷贝基β-globin基因序列的重组质粒作为标准品;收集无饲养层培养体系下人胚胎干细胞DNA样本,结合2个单独的Taqman探针实时荧光定量PCR对待测样本中线粒体NDl和核β-globin基因分别进行定量,从而对人胚胎干细胞线粒体DNA的含量进行了精确定量。结果提示,人胚胎干细胞线粒体DNA的平均拷贝数／细胞为1321±228。研究表明,该技术可对人胚胎干细胞线粒体DNA拷贝数进行准确的测定,为研究培养条件对人胚胎干细胞线粒体DNA拷贝数的影响及优化体外培养条件奠定了基础。     

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.     

The regulation of mitochondrial DNA copy number in glioblastoma cells

As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.     

Mouse models of mitochondrial DNA defects and their relevance for human disease

Qualitative and quantitative changes in mitochondrial DNA (mtDNA) have been shown to be common causes of inherited neurodegenerative and muscular diseases, and have also been implicated in ageing. These diseases can be caused by primary mtDNA mutations, or by defects in nuclear‐encoded mtDNA maintenance proteins that cause secondary mtDNA mutagenesis or instability. Furthermore, it has been proposed that mtDNA copy number affects cellular tolerance to environmental stress. However, the mechanisms that regulate mtDNA copy number and the tissue‐specific consequences of mtDNA mutations are largely unknown. As post‐mitotic tissues differ greatly from proliferating cultured cells in their need for mtDNA maintenance, and as most mitochondrial diseases affect post‐mitotic cell types, the mouse is an important model in which to study mtDNA defects. Here, we review recently developed mouse models, and their contribution to our knowledge of mtDNA maintenance and its role in disease.     

mtDNA拷贝数的生物学意义及其调控

线粒体是细胞能量和自由基代谢中心,并在细胞凋亡、钙调控、细胞周期和信号转导中发挥重要作用,维持线粒体功能正常对于细胞正常行使职能意义重大。线粒体的功能与线粒体DNA（mitochondrial DNA,mtDNA）的数量和质量紧密相关,mtDNA的数量即mtDNA拷贝数又受到mtDNA质量的影响,因此mtDNA拷贝数可作为线粒体功能的重要表征。mtDNA拷贝数变异引起线粒体功能紊乱,进而导致疾病发生。本文综述了mtDNA拷贝数变异与神经退行性疾病、心血管疾病、肿瘤等疾病的发生发展和个体衰老之间的关系,以及mtDNA复制转录相关因子、氧化应激、细胞自噬等因素介导mtDNA拷贝数变异的调控机制。以期为进一步深入探究mtDNA拷贝数调控的分子机制,以及未来治疗神经退行性疾病、肿瘤及延缓衰老等提供一定的理论基础。     

Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (P_trend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2^nd, 3^rd, 4^th, and 5^th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1^st quintile (P_trend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (P_trend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk.     

Analysis of mitochondrial DNA copy number variation in blood and tissue samples of metastatic breast cancer patients (A pilot study)

Changes in mitochondrial DNA (mt-DNA) copy number in blood/tissue have been linked to increased risk of several cancers; however, studies on their association in breast cancer is still lacking. In this pilot study, we investigated mt-DNA copy number variation in peripheral blood and tissue samples from metastatic breast cancer patients and compared their differences. For the study, peripheral blood samples from non-cancer individuals (control) and breast cancer patients, along with resected tissues from adjacent and tumor sites from same breast cancer patients were collected. Total genomic DNA was isolated and changes in mt-DNA copy number were measured by relative quantification using SYBR green based quantitative real time PCR method. Our results indicated a significant reduction in mt-DNA copy number in blood samples of breast cancer patients compared to control. However, a significantly higher mt-DNA copy number was observed in tumor tissue when compared with paired non tumor tissue. There was no significant difference in mt-DNA copy number between blood and adjacent tumor tissue samples of the breast cancer patients. Overall, our study reports for the first time a comparison of mt-DNA copy number in blood and paired tissue together and suggested that mt-DNA copy number is differentially regulated in blood and tumor tissues in breast cancer.     

Number matters: control of mammalian mitochondrial DNA copy number

Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.     

Oxidative stress-related alteration of the copy number of mitochondrial DNA in human leukocytes

The role of oxidative stress in the regulation of the copy number of mitochondrial DNA (mtDNA) in human leukocytes is unclear. In this study, we investigated the redox factors in plasma that may contribute to the alteration of mtDNA copy number in human leukocytes. A total of 156 healthy subjects of 25-80 years of age who exhibited no significant difference in the distribution of subpopulations of leukocytes in blood were recruited. Small-molecular-weight antioxidants and thiobarbituric acid reactive substances (TBARS) in plasma and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4,977bp deletion of mtDNA in leukocytes were determined. The mtDNA copy number in leukocytes was determined by real-time PCR. The results showed that the copy number of mtDNA in leukocytes was changed with age in a biphasic manner that fits in a positively quadratic regression model (P = 0.001). Retinol (P = 0.005), non-protein thiols (P = 0.001) and ferritin (P = 0.004) in plasma and total glutathione in erythrocytes (P = 0.046) were the significant redox factors that correlated with the mtDNA copy number in leukocytes in a positive manner. By contrast, alpha-tocopherol levels in plasma (P = 0.001) and erythrocytes (P = 0.033) were negatively correlated with the mtDNA copy number in leukocytes. Three oxidative indices including the incidence of 4,977 bp deletion of mtDNA (P = 0.016) and 8-OHdG content in leukocytes (P = 0.003) and TBARS in plasma (P = 0.001) were all positively correlated with the copy number of mtDNA in leukocytes. Taken these findings together, we suggest that the copy number of mtDNA in leukocytes is affected by oxidative stress in blood circulation elicited by the alteration of plasma antioxidants/prooxidants and oxidative damage to DNA.