目的 :建立抑制性差减文库为获得差异表达基因提供方法。方法 :提取正常组及卒中发作组大鼠全脑组织总RNA ,分离出mRNA。两组mRNA反转录合成cDMA。实验组称为tester,对照组称为driver。限制性内切酶RsaI消化产生平端。将testercDNA分成两份 ,分别连接不同的接头 (Adaptor) ,drivercDNA不连接接头。testercDNA连接好接头以后 ,再和driver进行两轮杂交。第一次PCR时仅有连接不同接头的双链cDNA分子可以呈指数扩增。然后采用巢式引物进行第二次PCR ,进一步减少PCR产物的背景 ,强化差异表达序列。结果 :总RMA经甲醛变性凝胶电泳紫外光下可见 2 8S、1 8S 条带清晰 ,2 8S:1 8S 比值约为 1 5 :1 ,未见降解。双链cDMA经RsaI消化 ,cDNA片段变小。已经扣除的cDMA ,GAPDH晚 5个～ 1 5个循环出现条带 ,说明差减文库得到了有效扣除。结论 :建立大鼠卒中发作及正常大鼠脑组织两个抑制性差减文库 ,为我们下一步研究卒中相关基因提供了方便 OBJECTIVE: To establish an inhibitory subtractive library to provide a method for obtaining differentially expressed genes. Methods: Total RNA was extracted from whole brain tissue of rats in normal group and stroke group, mRNA was isolated. Two sets of mRNA reverse transcription of cDMA. The experimental group is called tester and the control group is called driver. Restriction enzyme RsaI digestion produces blunt ends. The testercDNA is divided into two parts, each connected to a different adapter (adapter), drivercDNA connector is not connected. TestercDNA connected to the connector, and then the driver for two rounds of hybridization. At the first PCR, only double-stranded cDNA molecules linked to different linkers can be amplified exponentially. Then using nested primer for the second PCR further reduces the background of the PCR product and enhances the differentially expressed sequence. Results: The total RMA was observed by formaldehyde denaturing gel electrophoresis under ultraviolet light 28S, 1 8S band clear, 2 8S: 1 8S ratio of about 15: 1, no degradation. Double-stranded cDMA was digested with RsaI and the cDNA fragment became smaller. Bands of cDMA and GAPDH that have been deducted from 5 to 15 cycles late show that the subtractive library has been effectively deducted. CONCLUSION: Establishing two suppression subtractive libraries of stroke onset and normal rat brain tissue provides the convenience for our further study on stroke related genes