Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR.
Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR.
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DOI：
10.1186/1471-2229-10-146
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发表时间：
2010-07-15
影响因子：
5.3
通讯作者：
Kundu JK
中科院分区：
文献类型：
--
作者：
Jarosová J;Kundu JK
参考基因通常被用作相对量化目标基因的内源性归一化度量。然而，定量实时聚合酶链式反应(RT-qPCR)的适当应用需要使用其表达水平不受测试、一般生理条件或个体间变异性影响的参考基因。为此，在最重要的谷物(小麦、大麦和燕麦)的组织中研究了七个参考基因。采用不同参考基因的相对定量和绝对定量两种方法对燕麦上大麦黄矮病毒(BYDV)的滴度进行了测定，并对测定结果进行了比较。对180个健康、生理胁迫和病毒感染的谷类植物的组织中7个潜在的参考基因的表达进行了评估。用RT-qPCR对这些基因进行检测，并使用三种不同的方法(双向方差分析、GeNorm和NormFinder工具)根据其表达的稳定性进行排序。在大多数情况下，所有基因的表达都不依赖于非生物胁迫条件或病毒感染。这些基因在不同植物间的表达存在显著差异。甘油醛-3-磷酸脱氢酶(GAPDH)、β-微管蛋白(Tubb)和18S核糖体RNA(18S RRNA)一直是最稳定的三个基因。另一方面，大麦和燕麦样品中的延伸因子-1α(EF1a)、真核启动因子4a(EIF4A)和28S核糖体RNA(28S RRNA)以及小麦样品中的α-微管蛋白(Tuba)一直被列为不太可靠的对照。用三种不同的定量方法对两个燕麦品种的黄矮病病毒滴度进行了RT-qPCR测定。绝对定量与相对定量之间、GAPDH+Tubb+Tuba+18S rRNA定量与EF1A+eIF4A+28S rRNA定量之间均无显著差异。然而，单项检测结果之间存在差异。GAPDH、18S rRNA和Tubb的几何平均值适合大麦组织中BYDV定量的归一化。对于小麦和燕麦样品，4个基因的组合是必要的：小麦的GAPDH、18S rRNA、Tubb和eIF4A；燕麦的GAPDH、18S rRNA、Tubb和tuba。
Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR), however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in tissues of the most important cereals (wheat, barley and oats). Titre of Barley yellow dwarf virus (BYDV) was determined in oats using relative quantification with different reference genes and absolute quantification, and the results were compared. The expression of seven potential reference genes was evaluated in tissues of 180 healthy, physiologically stressed and virus-infected cereal plants. These genes were tested by RT-qPCR and ranked according to the stability of their expression using three different methods (two-way ANOVA, GeNorm and NormFinder tools). In most cases, the expression of all genes did not depend on abiotic stress conditions or virus infections. All the genes showed significant differences in expression among plant species. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUBB) and 18S ribosomal RNA (18S rRNA) always ranked as the three most stable genes. On the other hand, elongation factor-1 alpha (EF1A), eukaryotic initiation factor 4a (EIF4A), and 28S ribosomal RNA (28S rRNA) for barley and oat samples; and alpha-tubulin (TUBA) for wheat samples were consistently ranked as the less reliable controls. The BYDV titre was determined in two oat varieties by RT-qPCR using three different quantification approaches. There were no significant differences between the absolute and relative quantifications, or between quantification using GAPDH + TUBB + TUBA +18S rRNA and EF1A + EIF4A + 28S rRNA. However, there were discrepancies between the results of individual assays. The geometric average of GAPDH, 18S rRNA and TUBB is suitable for normalisation of BYDV quantification in barley tissues. For wheat and oat samples, a combination of four genes is necessary: GAPDH, 18S rRNA, TUBB and EIF4A for wheat; and GAPDH, 18S rRNA, TUBB and TUBA for oat is recommended.
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DOI：
10.1016/s0946-672x(03)80005-6
发表时间：
2003-01-01
影响因子：
3.5
作者：
Pfaffl, MW;Windisch, W
通讯作者：
Windisch, W
影响因子：
3.2
作者：
Balaji, B;Bucholtz, DB;Anderson, JM
通讯作者：
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影响因子：
4.2
作者：
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Shi, Wei Ming
影响因子：
11.2
作者：
Andersen, CL;Jensen, JL;Orntoft, TF
通讯作者：
Orntoft, TF
影响因子：
64.5
作者：
Chen, Brian E.;Kondo, Masahiro;Schmucker, Dietmar
通讯作者：
Schmucker, Dietmar