番茄种子RNA提取及快速检测qRT-PCR模板纯度的方法优化

The Optimization Method on RNA Extraction of Tomato Seed and Rapid Detection of Purity for qRT-PCR Template

番茄种子内富含多糖、贮藏蛋白、脂肪,同时还含有酚、酮等多种次生代谢物质,给提取高质量的RNA进行基因表达相关研究带来了的困难。本研究通过优化北京华越洋生物科技有限公司RNA提取试剂盒的操作流程,获得大量完整的RNA,并通过设计跨越内参基因(EF 1α)内含子的引物鉴定DNA的污染。结果表明,以水和RNA为模板没有扩增出片段,以种子DNA为模板扩增出包含内含子的441bp DNA片段,以cDNA为模板扩增出363bp的DNA片段,这说明cDNA没有被DNA污染。利用这种策略分析了种子萌发过程中2个基因的表达水平,基因表达结果证明了这是一种快速、有效的方法。另外,这种策略为植物转录组学的相关研究提供了参考方法。

In tomato seeds,there are lots of polysaccharides,protein and fat.In addition,it also contains phenol,ketones and other secondary metabolites.Therefore,it will bring great difficulty for many researches of gene expression on the extraction of high quality RNA.In our study,the intact RNA in the seed tissue can be obtained by improving the RNA extraction process of kit（Huayueyang Biotech Co.,Ltd）.Many laboratories have used RT-qPCR as a method for measuring genes expression in transcript levels because of its high precision and high sensitivity.The accuracy of RT-PCR gene expression analysis is affected by many factors,and DNA contamination is one of the main factors affecting the quality of the template.Therefore,it is the key to determine whether the RNA was contaminated with genomic DNA,primers which span a small intron of the house keeping gene EF 1were used.Amplification of cDNA samples yielded a gene-specific band（363bp）,while no product could be amplified from non-retrotranscribed RNA sample,indicating that the synthesized cDNA is not contaminated by genomic DNA.Through this strategy,expression levels of two genes involved in germination expression are determined.The results have indicated that it is a fast and effective method.In addition,this policy provides a reference method for plant transcriptomics study.