番茄miRNA的茎环qRT-PCR方法验证

Stem-Loop Real Time Quantitative PCR for Quantification of miRNA in Tomato

本研究采用SYBR Green染料的茎环q RT-PCR方法,以mi R159和mi R172为例,利用番茄叶片的总RNA作为模板,设计mi RNA的茎环引物、特异上游引物、通用下游引物,使用茎环荧光定量PCR法定量检测mi RNA表达量在番茄中的可行性。通过分析扩增曲线、熔解曲线,制作标准曲线,以及对PCR产物的克隆测序,结果表明该方法可以用于番茄mi RNA的定量检测,并且具敏感性高、特异性好、操作简单、节约成本、模板用量少的优点。Trizol法和试剂盒法提取的总RNA在后续实验中几乎没有差异,但Trizol法更为经济,有效率。Mi RNAs在植物的生长发育过程中起着重要的作用,本研究为番茄mi RNA的深入研究奠定了基础。

The study was designed to verify the effectiveness of stem-loop q RT-PCR method in tomato mi RNA quantitative detection. We took mi RNA159 and mi RNA172 as examples and designed mi RNA stem-loop primer,specific forward primer and universal primer and used SYBR Green as dye and total RNA as template. In this study,we analyzed q RT-PCR amplification curves,melting curves and standard curves. Then we cloned and sequenced PCR products. The results showed that this protocol can be used in quantitative detection of mi RNA in tomato with advantages of high sensitivity and specificity,easier operation,low concentration of template and cost-saving. There are almost no differences between total RNA trizol extraction method and the UNIQ-10 Column Trizol total RNA extraction kit(Shanghai Sangon) method in subsequent experiments,but Trizol method is more economical and efficient. MiRNAs play important roles in the growth and development of plants. This study might lay the foundation for further study of the mi RNA in tomato in the future.