Reverse transcription, quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression by transcript abundance. Expression of target genes is normalized to expression of stable reference genes to account for sample preparation variability. Thus, the identification and validation of stably expressed reference genes is crucial for making accurate, quantitative, statistical conclusions in gene expression studies. Traditional housekeeping genes identified decades ago based on high and relatively stable expression are often used, although many have shown these to not be valid, particularly in highly dynamic systems such as stem cell differentiation. In this study we outline a rational approach to identify stable reference genes valid throughout human pluripotent stem cell (hPSC) differentiation to hPSC-derived cardiomyocytes (hPSC-CMs). Several publicly available transcriptomic data sets were analyzed to identify genes with low variability in expression throughout differentiation. These putative novel reference genes were subsequently validated in RT-qPCR analyses to assess their stability under various perturbations, including maturation during extended culture, lactate purification, and various differentiation efficiencies. Expression in hPSC-CMs was also compared with whole human heart tissue. A core set of three novel reference genes (EDF1, DDB1, and ZNF384) exhibited robust stability across the conditions tested, whereas expression of the traditional housekeeping genes tested (ACTB, B2M, GAPDH, and RPL13A) varied significantly under these conditions.

中文翻译：

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多能干细胞衍生心肌细胞参考基因的合理、无偏选择

逆转录、定量聚合酶链反应 (RT-qPCR) 是一种通过转录丰度来量化基因表达的强大技术。将目标基因的表达标准化为稳定参考基因的表达，以说明样品制备的可变性。因此，稳定表达的参考基因的鉴定和验证对于在基因表达研究中做出准确、定量、统计的结论至关重要。几十年前基于高且相对稳定的表达确定的传统管家基因经常被使用，尽管许多人已经证明这些基因是无效的，特别是在干细胞分化等高动态系统中。在本研究中，我们概述了一种合理的方法来识别在人类多能干细胞 (hPSC) 分化为 hPSC 衍生心肌细胞 (hPSC-CMs) 期间有效的稳定参考基因。分析了几个公开可用的转录组数据集，以鉴定在整个分化过程中表达变异性低的基因。这些推定的新参考基因随后在 RT-qPCR 分析中得到验证，以评估它们在各种扰动下的稳定性，包括扩展培养过程中的成熟、乳酸纯化和各种分化效率。hPSC-CMs 中的表达也与整个人类心脏组织进行了比较。一组核心的三个新参考基因（分析了几个公开可用的转录组数据集，以鉴定在整个分化过程中表达变异性低的基因。这些推定的新参考基因随后在 RT-qPCR 分析中得到验证，以评估它们在各种扰动下的稳定性，包括扩展培养过程中的成熟、乳酸纯化和各种分化效率。hPSC-CMs 中的表达也与整个人类心脏组织进行了比较。一组核心的三个新参考基因（分析了几个公开可用的转录组数据集，以鉴定在整个分化过程中表达变异性低的基因。这些推定的新参考基因随后在 RT-qPCR 分析中得到验证，以评估它们在各种扰动下的稳定性，包括扩展培养过程中的成熟、乳酸纯化和各种分化效率。hPSC-CMs 中的表达也与整个人类心脏组织进行了比较。一组核心的三个新参考基因（EDF1、DDB1和ZNF384）在测试条件下表现出强大的稳定性，而测试的传统管家基因（ACTB、B2M、GAPDH和RPL13A）的表达在这些条件下显着变化。