Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.).
Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.).
复制标题
选择亚麻中定量基因表达归一化的参考基因（linum usitatissimum L.）。
DOI：
10.1186/1471-2229-10-71
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发表时间：
2010-04-19
影响因子：
5.3
通讯作者：
Neutelings G
中科院分区：
文献类型：
--
作者：
Huis R;Hawkins S;Neutelings G
实时定量聚合酶链式反应(qRT-PCR)是目前检测基因差异表达最准确的方法。这种方法依赖于识别统一表达的“管家基因”(HKGs)。广泛的转录数据挖掘和在不同模式植物中的实验验证表明，这些内源调控的可靠性可以受到植物种类、生长条件和所研究的器官/组织的影响。因此，在使用qRT-PCR研究差异基因表达之前，确定在每个生物系统中使用的最佳参考基因是很重要的。在本文中，我们评估了不同的候选HKGs在经济上重要的亚麻纤维和油料作物(亚麻L)的发育转录研究中的作用。为了定量研究20个不同的看家基因在不同发育阶段的亚麻根、茎内外组织、叶片和花中的表达水平，设计了特异的引物。在计算了聚合酶链式反应效率后，保留了13个HKG，并用计算机算法geNorm和NormFinder评估了它们的表达稳定性。根据geNorm，当考虑到所有研究样本时，2个转录延长因子(TEF)和1个泛素基因是使基因表达正常化所必需的。然而，只需要2个TEF就可以使其在茎组织中正常表达。相比之下，NormFinder发现，当所有样本被归类在一起时，以及当样本被分成不同的亚组时，甘油醛-3-磷酸脱氢酶(GADPH)是最稳定表达的基因。然后用qRT-PCR检测亚麻LuMYB1基因(与AtMYB59同源)的两个剪接变异体的相对表达水平。与外茎组织和其他标本相比，LuMYB1-1和LuMYB1-2在内茎组织中高表达。这一结果被geNorm指定的参考基因和NormFinder指定的参考基因所证实。使用两种不同的统计算法导致识别用于表达数据归一化的亚麻HKG的不同组合。尽管有这些差异，使用geNorm指定的参考基因和NormFinder指定的参考基因使我们能够准确地比较亚麻MYB基因在不同器官和组织中的表达水平。我们对合适的亚麻HKGs的鉴定和验证将有助于在这种具有重要经济价值的植物上进行未来的发育转录研究。
Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L). Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups. qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes. The use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.
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影响因子：
6.9
作者：
Gutierrez, Laurent;Conejero, Genevieve;Van Wuytswinkel, Olivier
通讯作者：
Van Wuytswinkel, Olivier
影响因子：
6.9
作者：
Bomal, Claude;Bedon, Frank;Caron, Sebastien;Mansfield, Shawn D.;Levasseur, Caroline;Cooke, Janice E. K.;Blais, Sylvie;Tremblay, Laurence;Morency, Marie-Josee;Pavy, Nathalie;Grima-Pettenati, Jacqueline;Seguin, Armand;MacKay, John
通讯作者：
MacKay, John
影响因子：
7.4
作者：
Bracha-Drori, Keren;Shichrur, Keren;Yalovsky, Shaul
通讯作者：
Yalovsky, Shaul
影响因子：
4.3
作者：
Day, A;Ruel, K;Chabbert, B
通讯作者：
Chabbert, B
影响因子：
5.3
作者：
Diretto, Gianfranco;Welsch, Ralf;Tavazza, Raffaela;Mourgues, Fabienne;Pizzichini, Daniele;Beyer, Peter;Giuliano, Giovanni
通讯作者：
Giuliano, Giovanni