Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.
Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.
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DOI：
10.1371/journal.pone.0027592
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Golding MC
中科院分区：
文献类型：
--
作者：
Veazey KJ;Golding MC
胚胎和组织特异性干细胞的分离和培养为研究驱动发育的分子过程提供了巨大的机会。为了深入了解支持哺乳动物胚胎发生的初始事件，已经衍生了来自植入前胚泡内存在的三种不同谱系中的每一种的多能干细胞。胚胎（ES）、滋养外胚层（TS）和胚外内胚层（XEN）干细胞具有其创始谱系的发育潜力，并且似乎利用不同的表观遗传方式来编程基因表达。然而，这些不同的细胞身份和表观遗传特性的基础仍然很难定义。定量逆转录-聚合酶链反应（qPCR）是一种强大而有效的手段，可以快速比较不同发育阶段和实验条件下的基因表达模式。然而，仔细的，经验性的选择适当的参考基因是必不可少的准确测量转录差异。在这里，我们报告了ES，TS和XEN干细胞之间的14个常用参考基因的定量和评价。这些包括：Actb、B2 m、Hsp 70、Gapdh、Gusb、H2 afz、Hk 2、Hprt、Pgk 1、Ppia、Rn 7sk、Sdha、Tbp和Ywhaz。利用三个独立的统计分析，我们确定Pgk 1，Sdha和Tbp作为这些干细胞类型之间最稳定的参考基因。此外，我们确定Sdha，Tbp和Ywhaz以及Ywhaz，Pgk 1和Hk 2作为三个最稳定的参考基因，通过胚胎干细胞和滋养外胚层干细胞的体外分化。了解这些不同的干细胞类型内控制细胞身份的转录和表观遗传调控机制，为控制胚胎发生和干细胞生物学的细胞过程提供了重要的见解。归一化定量RT-PCR测量使用几何平均CT值获得的确定的mRNA，提供了一种可靠的方法来评估不同模式的基因表达之间的三个创始干细胞谱系存在于哺乳动物植入前胚胎。
Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined. Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively. Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.
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