Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079, USA.
Biotechniques. 2009 Oct;47(4):867-9. doi: 10.2144/000113222.
Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences in the extraction efficiency of nDNA and mtDNA and variation in the ploidy of experimental samples. Here, we report that the ratio of mtDNA to nDNA varies in repeated DNA extractions but that PhiX174 DNA, added before DNA extraction, is extracted with a similar efficiency to mtDNA, making it a suitable alternative reference for quantifying mtDNA copy number.
实时定量 PCR 已成为分析和定量线粒体 DNA（mtDNA）拷贝数变化的常用方法，通常将核 DNA（nDNA）用作 mtDNA 丰度的内参。根据我们的经验，使用 nDNA 作为内参存在问题，这是因为 nDNA 和 mtDNA 的提取效率不同，以及实验样本的倍性存在差异。在这里，我们报告称，mtDNA 与 nDNA 的比值在重复 DNA 提取过程中会发生变化，但在 DNA 提取之前添加的 PhiX174 DNA 的提取效率与 mtDNA 相似，因此它是一种替代定量 mtDNA 拷贝数的合适内参。
