Division of Human Genetics, Departement of Pediatrics, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne, Switzerland.
Biochem Biophys Res Commun. 2012 Jul 6;423(3):441-7. doi: 10.1016/j.bbrc.2012.05.121. Epub 2012 Jun 5.
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.
实时 PCR（qPCR）是通过核与线粒体基因座的相对比较来定量线粒体 DNA（mtDNA）的首选方法。定量异常 mtDNA 含量表明存在线粒体疾病，且主要以组织特异性方式存在。因此，处理易降解的生物材料是不可避免的。我们建立了一种基于扩增子大小增加的串联 qPCR 检测方法，用于测量任何 DNA 样本的降解状态。通过这种方法，我们可以排除由于降解样品（例如，长的外切后时间、自溶过程、冻融循环）导致的错误 mtDNA 定量，并确保在未降解的患者样本中进行异常 DNA 含量测量（例如，耗竭）。通过在受控条件下使用超声处理和 DNA 酶 I 消化制备降解 DNA，我们表明错误的定量是由于核基因组和线粒体基因组的不同保存质量所致。这两种基因组的不同降解导致降解样品中 mtDNA 拷贝数的高估或低估。此外，由于对具有异常 mtDNA 含量的线粒体疾病的档案组织进行分析可以精确阐明其分子发病机制，我们比较了同一样本的新鲜冷冻（FF）和福尔马林固定石蜡包埋（FFPE）骨骼肌组织。通过外推核 DNA（λnDNA）和 mtDNA（λmtDNA）的测量衰减常数，我们提出了一种在未来可能纠正降解样品中测量值的方法。据我们所知，这是首次证明了这两个基因组的不同降解影响，并系统评估了 DNA 降解对 mtDNA 拷贝数定量的影响。
