Wang Zhaohai, Wang Ya, Yang Jing, Hu Keke, An Baoguang, Deng Xiaolong, Li Yangsheng
State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Hubei, 430072, People's Republic of China.
State Key Laboratory of Hybrid Rice, Key Laboratory for Research and Utilization of Heterosis in Indica Rice, Ministry of Agriculture, College of Life Sciences, Wuhan University, Hubei, 430072, People's Republic of China.
Appl Biochem Biotechnol. 2016 Jul;179(5):753-75. doi: 10.1007/s12010-016-2029-4. Epub 2016 Mar 3.
Stable and uniform expression of reference genes across samples plays a key role in accurate normalization of gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). For rice study, there is still a lack of validation and recommendation of appropriate reference genes with high stability depending on experimental conditions. Eleven candidate reference genes potentially owning high stability were evaluated by geNorm and NormFinder for their expression stability in 22 various experimental conditions. Best combinations of multiple reference genes were recommended depending on experimental conditions, and the holistic stability of reference genes was also evaluated. Reference genes would become more variable and thus needed to be critically selected in experimental groups of tissues, heat, 6-benzylamino purine, and drought, but they were comparatively stable under cold, wound, and ultraviolet-B stresses. Triosephosphate isomerase (TI), profilin-2 (Profilin-2), ubiquitin-conjugating enzyme E2 (UBC), endothelial differentiation factor (Edf), and ADP-ribosylation factor (ARF) were stable in most of our experimental conditions. No universal reference gene showed good stability in all experimental conditions. To get accurate expression result, suitable combination of multiple reference genes for a specific experimental condition would be a better choice. This study provided an application guideline to select stable reference genes for rice gene expression study.
跨样本的内参基因稳定且一致的表达在通过逆转录定量聚合酶链反应（RT-qPCR）进行基因表达的准确标准化中起着关键作用。对于水稻研究而言，仍缺乏依据实验条件对具有高稳定性的合适内参基因进行验证和推荐。通过geNorm和NormFinder评估了11个潜在具有高稳定性的候选内参基因在22种不同实验条件下的表达稳定性。依据实验条件推荐了多个内参基因的最佳组合，并且还评估了内参基因的整体稳定性。在组织、热、6-苄氨基嘌呤和干旱的实验组中，内参基因会变得更具变异性，因此需要谨慎选择，但在冷、伤口和紫外线-B胁迫下它们相对稳定。磷酸丙糖异构酶（TI）、肌动蛋白结合蛋白-2（Profilin-2）、泛素结合酶E2（UBC）、内皮分化因子（Edf）和ADP-核糖基化因子（ARF）在我们的大多数实验条件下都很稳定。没有通用的内参基因在所有实验条件下都表现出良好的稳定性。为了获得准确的表达结果，针对特定实验条件选择多个内参基因的合适组合将是更好的选择。本研究为水稻基因表达研究选择稳定的内参基因提供了应用指南。
