UMR INRA-USTL 1281 Stress Abiotiques et Différenciation des Végétaux Cultivés, Université Lille 1, Villeneuve d'Ascq, France.
BMC Plant Biol. 2010 Apr 19;10:71. doi: 10.1186/1471-2229-10-71.
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.
实时荧光定量 PCR（qRT-PCR）是目前检测差异基因表达最准确的方法。这种方法依赖于鉴定均匀表达的“管家基因”（HKG）。在不同模式植物中的广泛转录组数据挖掘和实验验证表明，这些内参的可靠性可能受到植物种类、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，确定每个生物系统中使用的最佳参考基因非常重要。在本文中，我们评估了经济上重要的亚麻纤维和油作物（Linum usitatissimum L）发育转录组研究中不同候选 HKG。
为了定量分析亚麻根、内部茎组织、外部茎组织、叶片和花在不同发育阶段的 20 种不同潜在管家基因的表达水平，设计了特异性引物。计算 PCR 效率后，保留了 13 个 HKG，并通过计算机算法 geNorm 和 NormFinder 评估其表达稳定性。根据 geNorm，当考虑所有研究样本时，2 个转录延伸因子（TEFs）和 1 个泛素基因是正常化基因表达所必需的。然而，仅当考虑茎组织时，需要 2 个 TEF 来正常化表达。相比之下，当将所有样本归为一组时，以及将样本分为不同的亚组时，NormFinder 将甘油醛-3-磷酸脱氢酶（GADPH）鉴定为最稳定表达的基因。然后使用 qRT-PCR 研究了亚麻 LuMYB1 基因（AtMYB59 的同源物）的两个剪接变体的相对表达水平。与外部茎组织和其他样本相比，LuMYB1-1 和 LuMYB1-2 在内部茎组织中高度表达。这一结果得到了 geNorm 指定和 NormFinder 指定参考基因的验证。
使用 2 种不同的统计算法会导致鉴定出不同的亚麻 HKG 组合用于表达数据归一化。尽管存在差异，但使用 geNorm 指定和 NormFinder 指定的参考基因使我们能够准确比较不同器官和组织中亚麻 MYB 基因的表达水平。我们对合适的亚麻 HKG 的鉴定和验证将有助于今后在这种经济上重要的植物中进行发育转录组研究。
