大黄鱼microRNA荧光定量PCR中内参基因的选择与评价

发布时间：2018-04-11 04:09

本文选题：大黄鱼 + microRNA　；参考：《核农学报》2017年07期

【摘要】：为筛选适用于大黄鱼miRNA研究的内参基因,选取饥饿试验组(EG)中饥饿21d及正常投喂组(CG,对照)的大黄鱼肌肉组织进行miRNA高通量测序,初步筛选得到稳定表达的6个miRNA:miR-23a-3p,miR-4508,miR-1961,miR-1297,miR-1956-3p和Let-7。采用实时荧光定量PCR法检测这6个miRNA和3个传统的内参基因(U6,5S和18S)在大黄鱼6种组织(肌肉、肝脏、肾、脾、脑和心脏)以及不同饥饿时间段的肌肉组织中的表达,并利用Ge Norm,Best Keeper以及Norm Finder对数据进行分析。结果表明,miRNA的表达稳定性优于传统内参基因。在CG组大黄鱼不同组织中,最稳定单内参基因是miR-23a-3p,最佳内参基因组合为miR-23a-3p和Let-7。在不同饥饿时间的EG组大黄鱼肌肉组织中,最稳定单内参基因是miR-23a-3p,最佳内参基因组合为Let-7和miR-1961。本试验结果为研究大黄鱼miRNA时内参基因的选择提供了参考。
[Abstract]:In order to screen the internal reference gene suitable for the miRNA study of large yellow croaker, the muscle tissues of large yellow croaker, which were starved for 21 days and fed on normal diet group, were selected for high-throughput miRNA sequencing. Six miRNAs, miRNA-miR-23a-3pmcmiR-4508miR-1961miR-1297miR-1956-3p and Let-7miR-1956-3p were obtained.The expression of these six miRNA and three traditional internal reference genes (U6N5S and 18s) in six tissues (muscle, liver, kidney, spleen, brain and heart) of large yellow croaker and the muscle tissues of different starvation periods were detected by real-time fluorescence quantitative PCR.The data are analyzed by GE Norm Best Keeper and Norm Finder.The results showed that the expression stability of miRNA was superior to that of traditional reference gene.The most stable single reference gene was miR-23a-3pin different tissues of large yellow croaker in CG group. The best combination of internal reference gene was miR-23a-3p and Let-7.The most stable single reference gene was miR-23a-3p in muscle tissue of large yellow croaker with different starvation time. The best combination of internal reference gene was Let-7 and miR-1961.The results of this study provided a reference for the selection of internal reference gene in miRNA of Pseudosciaena Crocea.
【作者单位】：宁波大学海洋学院;宁波大学浙江海洋高效健康养殖协同创新中心;
【基金】：浙江省自然科学基金(LY15C190005) 浙江省科技厅项目(2016C02055-7) 宁波市科技局项目(2015C110005)
【分类号】：Q78;S917.4

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