荧光标记mRNA差异显示技术

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３００ｂｐ￣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

FLUORESCENT mRNA DIFFERENTIAL DISPLAY TECHNIQUE

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.