Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions
Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions
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DOI：
10.1007/s00425-010-1158-1
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发表时间：
2010-06-01
期刊：
影响因子：
4.3
通讯作者：
Gonzalez-Verdejo, Clara I.
中科院分区：
文献类型：
--
作者：
Die, Jose V.;Roman, Belen;Gonzalez-Verdejo, Clara I.
逆转录实时定量聚合酶链反应是生物系统中基因表达的最准确测量方法。数据通过一个称为标准化的过程进行分析。内标对于确定不同样品中的相对基因表达是必不可少的。为此目的，基于它们在样品中的组成型表达来选择参考基因。目前，还没有在任何生物体中鉴定出在不同组织类型或疾病情况下普遍最佳的任何参考基因。我们的目标是测试豌豆的11个潜在参考的调节。其中包括8个常用候选人和3个新候选人。本分析涉及26个样本，包括不同的组织、处理和基因型。为了可靠的数据标准化，每个实验组中最合适的参考基因组合是用整个实验系列中五种更稳定表达的参考基因中的至少两种（即蛋白磷酸酶2A、β-微管蛋白、GH 720838、肌动蛋白和GH 720808）构建的。为了验证基因稳定性的测定，使用不同的归一化因子计算基因特异性变异。当使用最稳定的基因时，去除了最非特异性的变异，突出了在基因表达实验中适当选择内部对照的重要性。这里提出的参考基因集将提供有用的指导方针，作为参考基因选择的起点，在豌豆研究的条件下，而不是在这里测试。
Reverse transcription quantitative real-time polymerase chain reaction is the most accurate measure of gene expression in biological systems. The data are analyzed through a process called normalization. Internal standards are essential for determining the relative gene expression in different samples. For this purpose, reference genes are selected based on their constitutive expression across samples. At present, there has not yet been any reference gene identified in any organism that is universally optimal across different tissue types or disease situations. Our goal was to test the regulation of 11 potential references for pea. These included eight commonly used and three new candidates. Twenty-six samples, including different tissues, treatments and genotypes, were addressed in this analysis. For reliable data normalization, the most suitable combination of reference genes in each experimental set was constructed with at least two out the five more stably expressed references in the whole experimental series (i.e. protein phosphatase 2A, beta-tubulin, GH720838, actin and GH720808). To validate the determined measure of gene-stability, the gene-specific variation was calculated using different normalization factors. The most non-specific variation was removed when the most stable genes were used, highlighting the importance of the adequate choice of internal controls in gene expression experiments. The set of reference genes presented here will provide useful guidelines as starting point for reference gene selection in pea studies under conditions other than those tested here.