Key Lab of Rubber Biology, Ministry of Agriculture & Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan 571737, China.
Plant Sci. 2011 Aug;181(2):132-9. doi: 10.1016/j.plantsci.2011.04.014. Epub 2011 Apr 30.
Real-time RT-PCR (RT-qPCR) is a sensitive and precise method of quantifying gene expression, however, suitable reference genes are required. Here, a systematic reference gene screening was performed by RT-qPCR on 22 candidate genes in Hevea brasiliensis. Two ubiquitin-protein ligases (UBC2a and UBC4) were the most stable when all samples were analyzed together. A mitosis protein (YLS8) and a eukaryotic translation initiation factor (eIF1Aa) were the most stable in response to tapping. UBC2b and UBC1 were the most stable among different genotypes. UBC2b and a DEAD box RNA helicase (RH2b) were the most stable across individual trees. YLS8 and RH8 were most stably expressed in hormone-treated samples. Expression of the candidate reference genes varied significantly across different tissues, and at least four genes (RH2b, RH8, UBC2a and eIF2) were needed for expression normalization. In addition, examination of relative expression of a sucrose transporter HbSUT3 in different RNA samples demonstrated the importance of additional reference genes to ensure accurate quantitative expression analysis. Overall, our work serves as a guide for selection of reference genes in RT-qPCR gene expression studies in H. brasiliensis.
实时 RT-PCR（RT-qPCR）是一种定量基因表达的敏感且精确的方法，但是需要合适的参考基因。在这里，通过 RT-qPCR 对巴西橡胶树中的 22 个候选基因进行了系统的参考基因筛选。当一起分析所有样本时，两种泛素蛋白连接酶（UBC2a 和 UBC4）最稳定。在应对割胶时，一种有丝分裂蛋白（YLS8）和一种真核翻译起始因子（eIF1Aa）最稳定。UBC2b 和 UBC1 在不同基因型中最稳定。UBC2b 和 DEAD 盒 RNA 解旋酶（RH2b）在个体树之间最稳定。YLS8 和 RH8 在激素处理的样品中表达最稳定。候选参考基因在不同组织中的表达差异很大，至少需要四个基因（RH2b、RH8、UBC2a 和 eIF2）进行表达归一化。此外，在不同 RNA 样品中检测蔗糖转运蛋白 HbSUT3 的相对表达表明，需要额外的参考基因来确保准确的定量表达分析。总的来说，我们的工作为巴西橡胶树 RT-qPCR 基因表达研究中参考基因的选择提供了指导。
