Moraes G P, Benitez L C, do Amaral M N, Vighi I L, Auler P A, da Maia L C, Bianchi V J, Braga E J B
Departamento de Botânica, Universidade Federal de Pelotas, Pelotas, RS, Brasil
Departamento de Botânica, Universidade Federal de Pelotas, Pelotas, RS, Brasil.
Genet Mol Res. 2015 Mar 27;14(1):2384-98. doi: 10.4238/2015.March.27.24.
To obtain accurate and reliable results for the expression of genes of interest using quantitative real-time polymerase chain reaction (RT-qPCR) techniques, it is necessary to normalize the data by comparing them to constitutive genes that exhibit uniform expression levels under experimental conditions. In this study, the stability of expression was evaluated for the following ten candidate reference genes in rice leaves (Oryza sativa L.) from the BRS Bojuru and BRS Ligeirinho genotypes that were subjected to salt stress (150 mM): actin 11 (ACT11), beta-tubulin (β-TUB), eukaryote elongation factor 1-α (Eef-1), eukaryotic initiation factor 4-α (eIF-4-α), E2 ubiquitin-conjugating enzyme (UBC-E2), ubiquitin 5 (UBQ5), ubiquitin 10 (UBQ10), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TIP41-like, and cyclophilin. The stability of expression for the aforementioned genes was then compared to that of three LTP genes using UBQ10, Eef-1, and eIF-4-α as references. After analyzing the expression levels using analysis of variance tests, the results indicated that UBQ10 was the most stable in all treatments (M = 0.404 and SV = 0.327). Furthermore, the eIF-4-α, TIP41-like, and cyclophilin genes exhibited the highest total coefficient of variation (CV = 269, 169.2, 179.2, respectively), which signifies that they exhibited the least stable expression. The expression levels of each candidate gene (LTP7, LTP10, and LTP13) were in contrast to the reference genes. Therefore, we concluded that UBQ10 is the best reference gene for RT-qPCR reactions under the experimental conditions. The expression analysis of LTP7, LTP10, and LTP13 confirmed the importance of validating reference genes to achieve accurate RT-qPCR results.
为了使用定量实时聚合酶链反应（RT-qPCR）技术获得感兴趣基因表达的准确可靠结果，有必要通过将数据与在实验条件下表现出均匀表达水平的组成型基因进行比较来对数据进行标准化。在本研究中，评估了来自BRS Bojuru和BRS Ligeirinho基因型的水稻叶片（Oryza sativa L.）中以下十个候选参考基因在盐胁迫（150 mM）下的表达稳定性：肌动蛋白11（ACT11）、β-微管蛋白（β-TUB）、真核生物延伸因子1-α（Eef-1）、真核生物起始因子4-α（eIF-4-α）、E2泛素结合酶（UBC-E2）、泛素5（UBQ5）、泛素10（UBQ10）、甘油醛-3-磷酸脱氢酶（GAPDH）、TIP41样蛋白和亲环蛋白。然后以UBQ10、Eef-1和eIF-4-α为参考，将上述基因的表达稳定性与三个LTP基因的表达稳定性进行比较。在使用方差分析测试分析表达水平后，结果表明UBQ10在所有处理中最稳定（M = 0.404，SV = 0.327）。此外，eIF-4-α、TIP41样蛋白和亲环蛋白基因表现出最高的总变异系数（CV分别为269、169.2、179.2），这表明它们的表达最不稳定。每个候选基因（LTP7、LTP10和LTP13）的表达水平与参考基因相反。因此，我们得出结论，在实验条件下，UBQ10是RT-qPCR反应的最佳参考基因。LTP7、LTP10和LTP13的表达分析证实了验证参考基因以获得准确RT-qPCR结果的重要性。
