Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Food Quality and Safety of Jiangsu Province, State Key Laboratory Breeding Base, Nanjing 210014, China.
National University of Singapore (Suzhou) Research Institute, Department of Biological Sciences, National University of Singapore, Kent Ridge 117543, Singapore.
Genes (Basel). 2019 Nov 4;10(11):887. doi: 10.3390/genes10110887.
The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable reference genes are still very limited. is a destructive rice pest and it can transmit multiple viruses, including (RBSDV), (RSV), and (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in at different developmental stages, in different tissues, in RBSDV- or RSV-infected or in RBSDV-infected and ()-silenced . The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 () and are the most stably expressed genes in at different developmental stages. Alpha-1-tubulin () is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) . is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) , while beta-tubulin () is the most stably expressed reference gene in RBSDV-V and -silenced . The selected reference genes were further investigated during analyses of RBSDV and gene expression in different tissues from RBSDV-V or RBSDV-NV . The stably expressed reference genes identified in this study will benefit future gene function studies using .
反转录定量聚合酶链反应（RT-qPCR）已广泛用于确定（小褐飞虱）中的基因功能。选择合适的参考基因（s）用于 RT-qPCR 数据的归一化对于可靠的结果至关重要。迄今为止，关于合适参考基因鉴定的报告仍然非常有限。是一种破坏性的水稻害虫，它可以传播多种病毒，包括（RBSDV）、（RSV）和（MRDV），到全球许多重要的谷类作物。在本研究中，我们研究了 7 个选定候选参考基因在不同发育阶段、不同组织、RBSDV 或 RSV 感染的或 RBSDV 感染和（）沉默的中的稳定性。使用 5 种不同的方法分别分析代表单个候选基因的 RT-qPCR 数据：Delta Ct 方法、geNorm、NormFinder、BestKeeper 和 RefFinder 算法。根据使用 RefFinder 方法的综合分析，选择特定条件下最稳定的参考基因。核糖体蛋白 L5（）和是不同发育阶段中表达最稳定的基因。α-1-微管蛋白（）是 RBSDV 毒力（RBSDV-V）或非毒力（RBSDV-NV）不同组织中表达最稳定的参考基因。是 RBSDV-V 或 RSV 毒力（RSV-V）中表达最稳定的参考基因，而β-微管蛋白（）是 RBSDV-V 和 -沉默中表达最稳定的参考基因。在所选择的参考基因进一步研究了在不同组织中的 RBSDV 基因表达在 RBSDV-V 或 RBSDV-NV 中。本研究中鉴定的稳定表达的参考基因将有助于未来使用 进行基因功能研究。
