卒中大鼠脑组织抑制性差减文库的构建

Construction of suppression subtractive libraries of brain tissue from apoplectic stroke rats and normal rats

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 。

Objective:To construct suppression subtractive libraries for identifying differentially expressed genes Methods:Total RNA was extracted from the whole brain tissue of normal and apoplectic stroke rats,from which mRNA was isolated to be the samples with different origination The experiment group was chosen as the tester and the control group as the driver The double-stranded cDNA synthesized by tester mRNA and driver mRNA was digested with restriction enzyme(RasI)to produce flush end Tester cDNA was divided into two parts to ligate two different types of adaptors while driver cDNA did not ligate adaptors Hybridization was performed twice between tester cDNA and driver cDNA Only the cDNA with different adaptors were amplified in the first polymerase chain reaction(PCR) Meanwhile,we performed a second subtraction in reverse Results:Total RNA typically exhibited two bright bands in ultraviolet rays,which corresponded to ribosome 28s and 18s RNA with the intensity ratio of about 1 5/1 The double-stranded cDNA appeared as a smear on sepharose electrophoresis after RsaI digestion,the average cDNA size became smaller After subtraction,the abundance of GAPDH decreased significantly Conclusion:The procedure represents an efficient means of constructing suppression subtraction libraries,which must be useful for identifying stroke-associated genes