Grady John P, Murphy Julie L, Blakely Emma L, Haller Ronald G, Taylor Robert W, Turnbull Doug M, Tuppen Helen A L
Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom.
Department of Neurology, University of Texas Southwestern Medical Center and VA North Texas Medical Center, and Neuromuscular Center, Institute for Exercise and Environmental Medicine, Dallas, Texas, United States of America.
PLoS One. 2014 Dec 4;9(12):e114462. doi: 10.1371/journal.pone.0114462. eCollection 2014.
Accurate and reliable quantification of the abundance of mitochondrial DNA (mtDNA) molecules, both wild-type and those harbouring pathogenic mutations, is important not only for understanding the progression of mtDNA disease but also for evaluating novel therapeutic approaches. A clear understanding of the sensitivity of mtDNA measurement assays under different experimental conditions is therefore critical, however it is routinely lacking for most published mtDNA quantification assays. Here, we comprehensively assess the variability of two quantitative Taqman real-time PCR assays, a widely-applied MT-ND1/MT-ND4 multiplex mtDNA deletion assay and a recently developed MT-ND1/B2M singleplex mtDNA copy number assay, across a range of DNA concentrations and mtDNA deletion/copy number levels. Uniquely, we provide a specific guide detailing necessary numbers of sample and real-time PCR plate replicates for accurately and consistently determining a given difference in mtDNA deletion levels and copy number in homogenate skeletal muscle DNA.
准确且可靠地定量野生型以及携带致病性突变的线粒体DNA（mtDNA）分子的丰度，不仅对于理解线粒体DNA疾病的进展很重要，而且对于评估新的治疗方法也很重要。因此，清楚了解不同实验条件下线粒体DNA测量分析的灵敏度至关重要，然而，对于大多数已发表的线粒体DNA定量分析来说，这一点常常缺失。在此，我们全面评估了两种定量Taqman实时PCR分析方法的变异性，一种是广泛应用的MT-ND1/MT-ND4多重线粒体DNA缺失分析，另一种是最近开发的MT-ND1/B2M单重线粒体DNA拷贝数分析，评估范围涵盖一系列DNA浓度以及线粒体DNA缺失/拷贝数水平。独特的是，我们提供了一份详细指南，详述了为准确且一致地确定匀浆骨骼肌DNA中线粒体DNA缺失水平和拷贝数的给定差异所需的样本数量和实时PCR板重复次数。
