| 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 |
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| 引用本文： | 孙懿,曾思聪,胡亮,卢光琇,林戈.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[J].细胞生物学杂志,2012(7):685-689. |
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| 作者姓名： | 孙懿 曾思聪 胡亮 卢光琇 林戈 |
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| 作者单位： | [1]中南大学生殖与干细胞工程研究所,长沙410078 [2]人类干细胞国家工程研究中心,长沙410078 [3]卫生部人类干细胞与生殖工程重点实验室,长沙410078 |
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| 基金项目： | 国家自然科学基金（No.81101510）、湖南省自然科学基金青年基金（No.09JJ4009）、国家高技术研究发展计划（863）（No.2006AA02A102）、高等学校博士学科点专项科研基金新教师基金（No.200805331133）和中央高校基本科研业务费青年教师助推基金（No.201012200219）资助项目 |
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| 摘 要： | 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。
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| 关 键 词： | 人胚胎干细胞 线粒体DNA DNA拷贝数 |
A New Method for Precise Determination of Mitochondrial DNA Copies in Human Embryonic Stem Cells with Real-time PCR |
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| Sun Yi,Zeng Sicong,Hu Liang,Lu Guangxiu,Lin Ge.A New Method for Precise Determination of Mitochondrial DNA Copies in Human Embryonic Stem Cells with Real-time PCR[J].Chinese Journal of Cell Biology,2012(7):685-689. |
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| Authors: | Sun Yi Zeng Sicong Hu Liang Lu Guangxiu Lin Ge |
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| Institution: | . (lnstitute of Reproductive and Stem Cell Engineering, Central South University, Changsha 410078, China; National Research and Engineering Center of Human Stem Cells, Changsha 410078, China; Key Laboratory of Human Stem Cells and Reproductive Engineering, Ministry of Health, Changsha 410078, China) |
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| Abstract: | In this paper, we introduced a precise assay that determined mtDNA levels in human embryonic stem cells using Real-time PCR-based procedure. Human embryonic stem cells were cultured on feeder free system. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence including mitochondrial MT-NDl gene and single copy β-globin gene of nuclear. Copy number of mtDNA was determined by amplifying a short region of the MT-ND1 gene, and nuclear DNA content was determined by amplification of a segment of the single copy β-globin gene separately. Results showed that the copy number of mtDNA per diploid nuclear genome in human embryonic stem cells was 1 321±228. This study shows that PCR-based assay enables accurate determination of mtDNA relative to nuclear DNA, which lays the foundation for the study of culture conditions effect on mitochondrial DNA copies number in human embryonic stem cells and for the optimized culture conditions in vitro. |
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| Keywords: | human embryonic stem cell mitochondrial DNA DNA copy number |
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