【作者】 马芳； 刘哲； 黄进强； 康玉军； 王建福；

【Author】 Fang Ma;Zhe Liu;Jinqiang Huang;Yujun Kang;Jianfu Wang;College of Animal Science and Technology, Gansu Agricultural University;

【机构】 甘肃农业大学动物科学技术学院；

【摘要】 虹鳟鱼是一种主要的商业和冷水鱼,通常受到高温的影响。基因表达分析是了解虹鳟热应激机制的重要方法之一。定量实时PCR(RT-QPCR)是分析基因相对表达的有效方法。影响彩虹鳟RT-qPCR的因素很多,包括参考基因,但在热应激条件下,尚未发现虹鳟理想的参考基因。本研究基于RNA和miRNA高通量测序,从虹鳟肝脏和头肾中筛选出几种表达稳定的m RNA和microRNA,并筛选出四种常用参考基因和一种snRNA(U6)进行鉴定。用RT-qPCR方法检测虹鳟热应激时m RNA和miRNA的表达水平。应用geNorm、NormFinder、BestKeeper和ΔCt方法分析RT-qPCR数据,系统地评价这些候选m RNA和miRNA的表达稳定性。最后,采用几何平均数的方法对候选参考m RNAs和miRNAs的稳定性进行排序。最后结果显示,β-actin和EF1-α分别是肝和头肾最稳定的内参m RNA。Ssa-miR-26a-5p是肝脏中最稳定的内参miRNA,ssa-miR-462b-5p是头肾中最稳定的内参miRNA。本研究首次在热应激条件下对虹鳟RT-qPCR分析中合适的参考m RNA和miRNA进行筛选。更多还原

【Abstract】 Rainbow trout is a major commercial and cold-water fish, which is usually affected by temperatures. Gene expression analysis is one of the important methods to understand the mechanism of heat stress in rainbow trout. Quantitative real-time PCR(RT-qPCR) is an effective method to analyze the relative expression of genes. Several factors affect the RT-qPCR, including reference gene, however, no ideal reference genes have been reported for rainbow trout under heat stress. In this study, the expression stability several mRNAs and microRNAs(miRNAs) were selected from the liver and the head kidney of rainbow trout based on high-throughput data of RNA-seq and miRNA-seq, in addition, four commonly used reference genes and one snRNA(U6) were also selected to identify the ideal reference mRNAs and miRNAs for the expression levels of m RNA and miRNA by RT-qPCR in response to heat stress in rainbow trout. GeNorm, NormFinder, BestKeeper and comparative ΔCt method were used to analyze the RT-qPCR data to systematically assess the expression stability of these candidate mRNAs and miRNAs. Finally, stability of candidate reference mRNAs and miRNAs was ranked by taking geometric means. The final rankings showed that β-actin and EF1-α were the most stable expression of the reference m RNAs in liver and head kidney respectively. Ssa-miR-26 a-5 p was the most stable expression of the reference miRNAs in liver and ssa-miR-462 b-5 p was recommended for head kidney. The study is the first attempt to identify appropriate reference mRNAs and miRNAs in the analysis of RT-qPCR in rainbow trout under heat stress.更多还原