Validation of novel reference genes for RT-qPCR studies of gene expression in Xenopus tropicalis during embryonic and post-embryonic development
Validation of novel reference genes for RT-qPCR studies of gene expression in Xenopus tropicalis during embryonic and post-embryonic development
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DOI：
10.1002/dvdy.23972
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发表时间：
2013-06-01
影响因子：
2.5
通讯作者：
Pollet, Nicolas
中科院分区：
文献类型：
--
作者：
Dhorne-Pollet, Sophie;Thelie, Aurore;Pollet, Nicolas
背景：通过定量PCR准确解释转录组谱需要建立物种特异性标准。然而，用于评估非洲爪蟾和热带爪蟾RNA表达谱的参考基因的选择主要是基于历史原因，并且它们通常仅反映实验室的传统。结果如下：我们研究了10个基因（dicer 1、drosha、eef 1a 1、elavl 3、gsc、h4、odc 1、rpl 8、smn 2、tbp）的表达稳定性，其中8个基因通常用作已发表的RT-qPCR实验中的内部对照。我们定义了特异性引物对，并通过在热带爪蟾中进行RT-qPCR表达谱分析来评估其作为参考基因的适用性。在从卵到小蛙的15个发育阶段以及解剖的胚胎中测定了基因表达稳定性。结论：总的来说，我们确定了一组合格的参考基因的不同发育时期。我们建议从早期胚胎发育到变态末期使用dicer 1、drosha、eef 1a 1和smn 2。在胚胎发生早期，drosha、eef 1a 1、smn 2是合适的。对于整个胚后发育和包括前变态和变态顶极的变态阶段，我们推荐使用drosha和smn 2。这些参考基因应该证明它们在研究数据比较中的有用性。Developmental Dynamics 242：709-717，2013. (c)2013 Wiley Periodicals，Inc.
Background: Accurate interpretation of transcriptome profiling by quantitative PCR requires the establishment of species-specific standards. However, the selection of reference genes for assessing RNA expression profiles in Xenopus laevis and Xenopus tropicalis was mostly based on historical reasons and they often only reflect the traditions of a laboratory. Results: We investigated the expression stability of 10 genes (dicer1, drosha, eef1a1, elavl3, gsc, h4, odc1, rpl8, smn2, tbp), 8 of which are commonly used as internal controls in published RT-qPCR experiments. We defined specific primer pairs and evaluated their suitability as reference genes by performing RT-qPCR expression profiling in Xenopus tropicalis. Gene expression stability was assayed in a set of 15 developmental stages from the egg to the froglet, and in dissected embryos. Conclusions: Overall, we determined a set of qualified reference genes for distinct developmental periods. We recommend the use of dicer1, drosha, eef1a1, and smn2 from early embryonic development up to the end of metamorphosis. During early embryogenesis drosha, eef1a1, smn2 are suitable. For the whole post-embryonic development and for metamorphic stages including pro-metamorphosis and metamorphic climax, we recommend the use of drosha and smn2. These reference genes should prove their usefulness for data comparison across studies. Developmental Dynamics 242:709-717, 2013. (c) 2013 Wiley Periodicals, Inc.