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A New Method for Precise Determination of Mitochondrial DNA Copies in Human Embryonic Stem Cells with Real-time PCR
Sun Yi1,2,3, Zeng Sicong1,2,3, Hu Liang1,2,3, Lu Guangxiu1,2,3, Lin Ge1,2,3*
1Institute of Reproductive and Stem Cell Engineering, Central South University, Changsha 410078, China; 2National Research and Engineering Center of Human Stem Cells, Changsha 410078, China; 3Key Laboratory of Human Stem C
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-ND1 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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