棉花miRNA表达量检测方法Stem-loop RT-PCR的建立

Development of Stem-loop RT-PCR for Analysis of Cotton miRNA Level

为建立一套鉴定棉花miRNA表达丰度的分子技术,优化了Stem-loop RT-PCR方法,并通过分析棉花3个miRNA的表达谱来评价分析该技术体系的特性。首先设计3条引物(茎环特异性反转录引物、正向引物和通用反向引物),通过cDNA和RNA梯度稀释,分析引物的扩增效率和检测灵敏度。结果表明,Stem-loop RT-PCR方法中设计的引物具有较高的扩增效率和检测灵敏度,miR156和miR159扩增效率分别为102.0%和103.6%;并对不同miRNA分析其总RNA检测灵敏度,miR156和miR159的总RNA检测灵敏度差异较大,分别为20 ng和2 pg。同时,应用建立的棉花Stem-loop RT-PCR方法,成功地分析了Gh-miR156、Gh-miR159和Gh-miR5658在根茎叶中的表达量。由此可见,棉花Stem-loop RT-PCR方法具有灵敏度高、特异性强、成本较低和适用于一般实验室等优点,为棉花等植物miRNA相关研究提供了技术保证,加快了miRNA及其调控基因功能的研究步伐。

miRNA functions are involved in plant growth and defense, but simple and highly effective approaches for analyzing miRNA level still need to exploit. In this study, we developed a Stem-loop RT-PCR in cotton to analyze the contents of Gh-miR156, Gh-miR159 and Gh-miR5658. The three special primers were designed, including stem-loop primer, Forward primer and Universal reverse primer. Based on analysis of RT-qPCR Ct values of 10 - fold diluted eDNA, the two sets of primers possessed highly amplifying effective for monitoring miR156 and miR159 levels, reached 102.0% and 103.6% , respectively. The sensitivity assays of Stem-loop RT-PCR showed that the limited contents of total RNA for monitoringmiR156 and miR159 were obviously different, which was 20 ng and 2 pg, respectively. Moreover, the expression levels of Gh-miR156, Gh-miR159 and Gh-miR5658 in cotton root, stem and leaf could be well tested by this Stem-loop RT-PCR technique. These results suggested that Stem-loop RT-PCR is well fit for analyzing cotton miRNA level with simple and highly effective potential, which facilitates in plant gene function analysis, especially in miRNA regulation.