荧光标记mRNA差异显示技术

FLUORESCENT mRNA DIFFERENTIAL DISPLAY TECHNIQUE

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 0bp～ 2 0kb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。

Aim: To apply fluorescent mRNA differential display technique.Methods: Total RNA samples were extracted from human monocyte line U937 treatcd/untreated with IFN and LPS, and were used as templates in differential display PCR. The anchored primers used were labeled with the fluorescent tag. After running on 5.6% denaturing PAGE gel, differentially expressed bands were excised and recovered, and finally reamplified. Results: Three tested samples all showed amplified bands differed from 300 bp to 2.0 kb, the bands were bright and clear, the background was low. Both yes/no changes and upregulated/downregulated happenings were shown simultaneously. The reamplification bands were sharp and pure. Conclusion: We have successfully practiced fluorescent differential display technique in our lab. It is a fast, safe and cost effective method used to sereen unknown expressed genes.