mRNA差异显示方法研究进展.docVIP

目的：应用荧光标记的mBNA差异显示技术。 方法：提 取未经过／经过IFN、LPS处理的三组人单核细胞系U937的总RNA并以此为模板，采用荧光标 记的锚定引物，通过逆转录、差异显示PCR反应，经5．6％变性聚丙烯酰胺凝胶电泳分离差 异条带，回收后将其再扩增。结果：三组样本的 DD-PCR产物电泳显示长30 0 bp～2.0 kb不等的扩增片段，条带清晰、明亮，背景低，各样本相互间的差异不仅呈有 无的变化，亦表现出很多强弱改变；再扩增条带锐利、单一。结论：在本试 验室成功应用了荧光标记差异显示技术，可快速、敏感、经济有效地筛选各种未知的表达基 因。　　中图分类号：　Q781文献标识码： B　　文章编号：1000-6834(200 0)04-0373-04FLUORESCENT mRNA DIFFERENTIAL DISPLAY TECHNIQUEWANG Gang-shi　　（Department of Gastroenterology General Hospital of Chinese PLA , Beijing 100853;）　　WANG Meng-wei　　（Department of Gastroenterology General Hospital of Chinese PLA , Beijing 100853;）　　YOU Wei-di　　（Department of Gastroenterology General Hospital of Chinese PLA , Beijing 100853;）　　WANG Hong-fan g　　（ Institute of Zoology,Chinese Academy of Science, Beijing 100080）　　FENG Mei-fu　　（ Institute of Zoology,Chinese Academy of Science, Beijing 100080）　　ABSTRACT　　Aim: To apply fluorescent mRNA differential display technique.Met hods: Total RNA samples were extracted from human monocyte line U937 treat cd/untreated with IFN and LPS, and were used as templates in differential displa y PCR. The anchored primers used were labeled with the fluorescent tag. After ru nning on 5.6% denaturing PAGE gel, differentially expressed bands were excised a nd recovered, and finally reamplified. Results: Three tested samples all showed amplified bands differed from 300 bp to 2.0 kb, the bands were brigh t and clear, the background was low. Both yes/no changes and upregulated/downreg ulated happenings were shown simultaneously. The reamplification bands were shar p and pure. Conclusion: We have successfully practiced fluorescent differential display technique in our lab. It is a fast, safe and cost-effecti ve method used to sereen unknown expressed genes.　　KEY WORDS:　differential display;　RNA;　messenger ;　fluorescence　　mRNA差异显示技术(differential display，DD)是用于研究基因的差异表达的新方法。该技术自1992年［1］]被首次报道后，即以其不可替代的优势被广泛应用于生物医学 领域。在应用过程中不断得到改进［2］，并产生了诸多衍生技术如RPA(RNA finger printing by arbitrarily primed PCR)、GDD(ge