2014, 21(5): 920-928. doi: 10.3724/SP.J.1118.2014.00920
关键词： 线粒体DNA 含量 , 实时荧光定量PCR , 半滑舌鳎 , 优化
Keywords： mitochondrial DNA content , real-time quantitative PCR , Cynoglossus semilaevis , optimization
本研究旨在应用实时荧光定量PCR 技术, 建立半滑舌鳎(Cynoglossus semilaevis)线粒体DNA(mtDNA)含量测定方法并进行优化。通过质粒标准品的线性化处理、模板DNA 的酶切和超声处理、mtDNA 和核DNA(nDNA)基因引物的筛选实验, 建立半滑舌鳎mtDNA 含量测定方法。结果表明, 质粒标准品的构象对荧光定量PCR 标准曲线影响较大, 线性化的质粒更适合用作标准品;酚-氯仿方法提取的模板DNA 适用于mtDNA 含量测定, 无需进行预处理;用D-loop 和ND1 这2 对引物所得的拷贝数较小且结果一致, 适用于mtDNA 拷贝数的测定;以不同核基因为参照所得的mtDNA 含量可能存在差异, 当以单拷贝核基因ENC1 和MYH6 为参照时, 可以计算出单个细胞中mtDNA 含量, 若以多拷贝基因GAPDH 为参照, mtDNA 含量测定值则较小。采用本方法分别对半滑舌鳎肝、肾、脾和肌肉组织的mtDNA 含量进行重复性检测实验, 结果表明, 相同组织的mtDNA 含量显示出良好的重复性(P>0.05),而不同组织中mtDNA 含量具有差异性, 可见该方法稳定可靠, 能为海洋鱼类mtDNA 含量检测提供借鉴。
Mitochondrial DNA (mtDNA) content is typically estimated as the copy number ratio of mtDNA to nuclear DNA(nDNA). However, the accuracy of mtDNA content measurement is affected by many factors, including the conformation of plasmid standards and the DNA template, the coexistence of mtDNA pseudogenes in the nuclear genome, and selection of both mtDNA and nDNA primer-pairs. To minimize the influence of these factors, an optimized method to quantify the mtDNA content in different tissues of Cynoglossus semilaevis was established using real-time quantitative PCR (RT-qPCR). First, two sets of candidate standards (the circular and linear plasmid) and three sets of DNA templates (enzyme digested, ultrasonic treated, and untreated) were prepared to evaluate the influence of the DNA template conformation. Additionally, four mtDNA and three nDNA primer pairs were also tested to determine their adequacy for the qRT-PCR analysis. The linear plasmid standard was more appropriate than the circular one because the super helical structure of the circular plasmid caused significant overestimation in RT-qPCR. There was no significant difference in the estimates of mtDNA content resulting from different DNA templates, suggesting that the DNA extracted by phenol-chloroform is suitable without any pre-treatment for extraction. The D-loop and ND1 primers yielded the same copy number, which was also the lowest among all the mtDNA primer pairs. The copy numbers for ATP6 and COII were 3.5 and 1.5 times higher than those from D-loop and ND1, respectively. The higher copy number of ATP6 and COII may be related to the co-amplification of homologous pseudogenes in the nuclear genome. Single copy nDNA loci ENC1 and MYH6 can be used as references for detecting cell numbers of diploids, and the precise mtDNA content per cell can be calculated using the formula: mtDNA content = 2×mtDNA copy number/nDNA copy number. In contrast, the mtDNA content value was lower when using the multicopy nDNA gene locus GAPDH as a reference. To evaluate the accuracy and stability of this optimized method, we measured the mtDNA content in four tissues (liver, kidney, spleen, and muscle) of C. semilaevis. D-loop and ENC1 primer pairs were chosen for the RT-qPCR, and the mtDNA content per cell was estimated using the method established in this study. There was no significant difference between triplicate repeats in each tissue (P>0.05), which suggests that the method has excellent repeatability. Furthermore, there was a significant difference in mtDNA content among the different tissues: 244–255,156–172, 97–107, 86–89 copies per cell were detected in liver, muscle, kidney, and spleen, respectively.
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