EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.
EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.
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DOI：
10.1186/1471-2199-11-61
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发表时间：
2010-08-17
影响因子：
--
通讯作者：
Schiller PC
中科院分区：
文献类型：
--
作者：
Curtis KM;Gomez LA;Rios C;Garbayo E;Raval AP;Perez-Pinzon MA;Schiller PC
RT-qPCR 分析是间充质基质细胞 (MSC) 研究领域广泛使用的 mRNA 表达分析方法。由于 RT-qPCR 数据标准化和分析方法不同，体外和体内 MSC 研究之间的比较具有挑战性。因此，本研究重点关注假定的管家基因，与 MIAMI 和 RS-1 细胞等更同质的 MSC 群体相比，用于异质市售人类 MSC 之间 RT-qPCR 数据标准化。八个基因包括； ACTB、B2M、EF1α、GAPDH、RPL13a、YWHAZ、UBC 和 HPRT1 根据其表达水平和变异性被测试为可能的管家基因。 EF1α 和 RPL13a 经验证可用于 MIAMI 细胞在不同氧张力下的扩增、内皮分化、神经前体富集过程中的 RT-qPCR 分析，以及与 RS-1 细胞和市售 MSC 的比较。 RPL13a 和 YWHAZ 被验证为局灶性缺血动物模型中 MIAMI 细胞跨物种分析的标准化基因。 GAPDH 是 MSC 研究领域用于 RT-qPCR 数据标准化最常见的管家基因之一，被发现具有最高的变异性，被认为不适合 RT-qPCR 数据标准化。为了对异质 MSC 群体以及世界各地不同实验室使用的 MSC 等成体干细胞进行比较，拥有一套标准化、可重复的管家基因用于 RT-qPCR 分析非常重要。在本研究中，我们证明 EF1α、RPL13a 和 YWHAZ 是适合在体外表征和分化以及全脑缺血动物模型中进行 RT-qPCR 分析和比较几种人类 MSC 来源的基因。这将允许对多个 MSC 群体进行比较 RT-qPCR 分析，目标是未来用于疾病动物模型以及组织修复。
RT-qPCR analysis is a widely used method for the analysis of mRNA expression throughout the field of mesenchymal stromal cell (MSC) research. Comparison between MSC studies, both in vitro and in vivo, are challenging due to the varied methods of RT-qPCR data normalization and analysis. Therefore, this study focuses on putative housekeeping genes for the normalization of RT-qPCR data between heterogeneous commercially available human MSC, compared with more homogeneous populations of MSC such as MIAMI and RS-1 cells. Eight genes including; ACTB, B2M, EF1α, GAPDH, RPL13a, YWHAZ, UBC and HPRT1 were tested as possible housekeeping genes based on their expression level and variability. EF1α and RPL13a were validated for RT-qPCR analysis of MIAMI cells during expansion in varied oxygen tensions, endothelial differentiation, neural precursor enrichment, and during the comparison with RS-1 cells and commercially available MSC. RPL13a and YWHAZ were validated as normalization genes for the cross-species analysis of MIAMI cells in an animal model of focal ischemia. GAPDH, which is one of the most common housekeeping genes used for the normalization of RT-qPCR data in the field of MSC research, was found to have the highest variability and deemed not suitable for normalization of RT-qPCR data. In order to make comparisons between heterogeneous MSC populations, as well as adult stem cell like MSC which are used in different laboratories throughout the world, it is important to have a standardized, reproducible set of housekeeping genes for RT-qPCR analysis. In this study we demonstrate that EF1α, RPL13a and YWHAZ are suitable genes for the RT-qPCR analysis and comparison of several sources of human MSC during in vitro characterization and differentiation as well as in an ex vivo animal model of global cerebral ischemia. This will allow for the comparative RT-qPCR analysis of multiple MSC populations with the goal of future use in animal models of disease as well as tissue repair.
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影响因子：
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通讯作者：
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