Rational, Unbiased Selection of Reference Genes for Pluripotent Stem Cell-Derived Cardiomyocytes
Rational, Unbiased Selection of Reference Genes for Pluripotent Stem Cell-Derived Cardiomyocytes
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多能干细胞衍生心肌细胞参考基因的合理、公正选择
DOI：
10.1089/ten.tec.2021.0023
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Palecek, Sean P.
中科院分区：
文献类型：
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作者：
Simmons, Aaron D.;Palecek, Sean P.
逆转录定量聚合酶链反应（RT-qPCR）是一种通过转录本丰度定量基因表达的强大技术。将靶基因的表达相对于稳定参比基因的表达进行标准化，以考虑样品制备的可变性。因此，鉴定和验证稳定表达的参考基因对于在基因表达研究中做出准确、定量、统计学结论至关重要。几十年前基于高和相对稳定的表达鉴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，特别是在高度动态的系统中，如干细胞分化。在这项研究中，我们概述了一个合理的方法来确定稳定的参考基因有效的整个人类多能干细胞（hPSC）分化为hPSC衍生的心肌细胞（hPSC-CM）。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。随后在RT-qPCR分析中验证这些推定的新型参考基因，以评估它们在各种扰动下的稳定性，包括延长培养期间的成熟、乳酸纯化和各种分化效率。还将hPSC-CM中的表达与整个人心脏组织进行了比较。三个新参考基因的核心组（EDF 1、DDB 1和ZNF 384）在测试的条件下表现出稳健的稳定性，而测试的传统管家基因的表达（ACTB、B2M、GAPDH、和RPL 13 A）在这些条件下变化显著。影响声明本文提出了一种无偏的方法，用于选择和验证新的参考基因，用于真实的-使用来自RNA测序数据集的数据进行时间定量聚合酶链反应归一化。该方法比常用的参考基因鉴定出更稳健和稳定的参考基因，用于人多能干细胞向心肌细胞分化过程中的基因表达研究。这项研究还提供了一个路线图，用于确定参考基因，以评估其他动态细胞过程中的基因表达，包括干细胞分化为其他细胞类型。
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, andZNF384) exhibited robust stability across the conditions tested, whereas expression of the traditional housekeeping genes tested (ACTB,B2M,GAPDH, andRPL13A) varied significantly under these conditions.Impact statementThis article presents an unbiased method for the selection and validation of novel reference genes for real-time quantitative polymerase chain reaction normalization using data from RNA sequencing datasets. This method identified more robust and stable reference genes for gene expression studies during human pluripotent stem cell differentiation to cardiomyocytes than commonly used reference genes. This study also provides a roadmap for identifying reference genes for assessing gene expression during other dynamic cellular processes, including stem cell differentiation to other cell types.