Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process.
Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process.
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DOI：
10.1186/1471-2229-8-131
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发表时间：
2008-12-22
影响因子：
5.3
通讯作者：
Perez, Jose A.
中科院分区：
文献类型：
--
作者：
Exposito-Rodriguez, Marino;Borges, Andres A.;Borges-Perez, Andres;Perez, Jose A.
基因表达模式的阐明有助于更好地理解生物过程。实时定量RT-PCR已成为深入研究基因表达的标准方法。一个具有生物学意义的目标信使核糖核酸数量的报告需要准确和可靠的标准化，以便识别真正的基因特异性变异。归一化的目的是控制几个变量，如起始材料的不同数量和质量，从RNA到cDNA逆转录的不同酶效率，或组织或细胞之间整体转录活性的差异。管家基因作为内源控制的有效性取决于其在被分析样本小组中的表达水平的稳定性。在本报告中，我们描述了首次对番茄发育过程中潜在的内部控制进行系统评估，以确定哪些是实时RT-PCR最可靠的转录本。在这项研究中，我们评估了7个传统看家基因和4个新的看家基因在27个样本中的表达稳定性，这些样本代表了番茄不同发育阶段的不同组织和器官。首先，我们设计、测试和优化了实时荧光定量RT-PCR的扩增引物。然后，根据不同的统计过程，用三种互补的方法对每个候选基因的表达数据进行评估。我们的分析表明，SGN-U314153(CAC)、SGN-U321250(TIP41)、SGN-U346908(“表达”)和SGN-U316474(SAD)基因在番茄发育研究中提供了较好的转录标准化。我们推荐这些特别稳定的看家基因的不同组合，以适应不同发育系列的正常化，包括完整的番茄发育过程。这项工作是为定量实时RT-PCR研究番茄发育过程中的基因表达选择最佳内源对照的第一次努力。从我们的研究中，出现了一个控制基因的工具包，它在表达稳定性方面优于传统基因。
The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process. From our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability.
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