Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice
Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice
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公共转录组数据库辅助选择和验证水稻 qRT-PCR 的可靠参考基因
DOI：
10.1007/s11427-019-1553-5
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Chen Letian
中科院分区：
文献类型：
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作者：
Zhao Zhe;Zhang Zixu;Ding Zhi;Meng Hengjun;Shen Rongxin;Tang Huiwu;Liu Yao-Guang;Chen Letian
准确的定量逆转录聚合酶链式反应(qRT-PCR)要求可靠的参考基因在不同组织和发育阶段表达不同。然而，目前用于水稻qRT-PCR的可靠参考基因还很少。在这里，我们建立了一种有效的策略来识别新的参考基因(NRGs)，以便可靠地标准化水稻不同器官和发育阶段的qRT-PCR数据。我们利用水稻数据库的信息共享区选择了候选NRGs，并在水稻表达谱数据库(RiceXPro)数据中证实了它们的表达。来自RiceXPro数据的热图中的低变异(2.5周期量化)跨组织和发育阶段的基因被认为是稳定的NRGs。为了验证这一策略，我们选择了11个候选NRGs，用5个程序计算了它们在不同时空条件下的表达稳定性，并将这些基因与5个已建立的参考基因(ERGs)进行了比较。只有1个ERG(UBQ5)是可靠的，10个候选NRG比其余4个ERG更稳定。因此，公共转录数据库对识别非RGs很有用。我们选择了UFM1结合酶1的同源基因UFC1和粘附素FhaB的同源基因FhaB进行水稻QRT-PCR分析，它们的同源基因可能适用于其他单子叶植物。
Accurate quantitative reverse transcription PCR (qRT-PCR) requires reliable reference genes whose expression does not vary in different tissues and developmental stages. However, few reliable reference genes are available for qRT-PCR in rice (Oryza sativa). Here, we established an effective strategy for identifying novel reference genes (NRGs) for reliable normalization of qRT-PCR data in various rice organs and developmental stages. We selected candidate NRGs using the Information Commons for Rice Database and confirmed their expression in Rice Expression Profile Database (RiceXPro) data. Genes with low variation (<2.5 cycle quantification) across tissues and developmental stages, and little fluctuation in expression in heatmaps from RiceXPro data were considered stable NRGs. To validate this strategy, we selected 11 candidate NRGs and calculated their expression stability in different spatio-temporal conditions using five programs, and compared these genes with five established reference genes (ERGs). Only one of the ERGs (UBQ5) was reliable and 10 of the candidate NRGs were more stable than the four remaining ERGs. Therefore, public transcriptomic databases are useful for identifying NRGs. We selected two NRGs,UFC1(Homolog of UFM1-Conjugating Enzyme 1) andFhaB(Homolog of Adhesin FhaB) for qRT-PCR analysis in rice; their homologs might be suitable for other monocot plants.