RT-qPCR analyses on the osteogenic differentiation from human iPS cells: an investigation of reference genes
RT-qPCR analyses on the osteogenic differentiation from human iPS cells: an investigation of reference genes
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DOI：
10.1038/s41598-020-68752-2
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发表时间：
2020-07-16
影响因子：
4.6
通讯作者：
Tanaka, Yasuhito
中科院分区：
文献类型：
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作者：
Okamura, Kensuke;Inagaki, Yusuke;Tanaka, Yasuhito
逆转录定量PCR（RT-qPCR）用于定量基因表达，需要用参考基因进行标准化。我们试图确定最适合于从人诱导多能干细胞诱导成骨分化的实验的参考基因。在未分化维持培养基中培养，汇合后，在成骨分化培养基中进一步培养28天。对未分化标志物、成骨细胞和骨细胞分化标志物以及参考候选基因进行RT-qPCR。使用四种算法对每个参考候选基因的表达稳定性进行排序。一般排名确定TATA盒结合蛋白放在首位，其次是转铁蛋白受体，核糖体蛋白大P0，最后，β-2-微球蛋白，这是最不稳定的。有趣的是，普遍使用的GAPDH和ACTB被发现不适合。我们的研究结果强烈表明，需要评估每个实验的参考候选基因的表达稳定性。
Reverse transcription quantitative PCR (RT-qPCR) is used to quantify gene expression and require standardization with reference genes. We sought to identify the reference genes best suited for experiments that induce osteogenic differentiation from human induced pluripotent stem cells. They were cultured in an undifferentiated maintenance medium and after confluence, further cultured in an osteogenic differentiation medium for 28 days. RT-qPCR was performed on undifferentiation markers, osteoblast and osteocyte differentiation markers, and reference gene candidates. The expression stability of each reference gene candidate was ranked using four algorithms. General rankings identified TATA box binding protein in the first place, followed by transferrin receptor, ribosomal protein large P0, and finally, beta-2-microglobulin, which was revealed as the least stable. Interestingly, universally used GAPDH and ACTB were found to be unsuitable. Our findings strongly suggest a need to evaluate the expression stability of reference gene candidates for each experiment.