大鼠脑卒中相关基因的抑制性差减杂交文库构建

目的建立寒冷加高盐刺激大鼠脑卒中的大脑组织差异cDNA文库。 方法正常Wistar大鼠随机分为寒冷加高盐刺激的实验组和不予任何处理的对照组。取实验组发生脑卒中和对照组的大脑组织。提取两组组织的mRNA，按照抑制性差减杂交试剂盒说明进行实验。在三个水平设定了检验差减效率的实验。首先是使用试剂盒提供的骨骼肌mRNA加入外源性PhiX174（HaeⅢ）作为阳性实验组与我们的实验样品进行平行差减，用来证明系统正常。其次是采用PCR扩增看家基因GPDH的方法观察本次实验的差减效果。最后在两库随机各挑取288个克隆测序，比较相同序列的重复情况。 结果差减各过程符合理论质量要求，分别得到一个脑卒中特异表达基因的片段库和一个正常特异表达基因片段库。卒中库的理论容量是7.1×10

Construction of Differential Library of Brain in A Rat Model of Stroke

Objective to construct differential libraries of brain in a rat mode l of stroke, which is induced by combining cold and high salt stimulation. Methods Normal Wistar rats were divided randomly into control and test group. The test group was stimulated with high salt and cold environment while the control group was reared without any treatment. Cereb rums of stroke rats in test group and of normal rats in control group were collected. Message R NA was extracted from those cerebrums. Suppression subtractive hybridization was conducted accord ing to manufacture's manual. Subtractive efficiency was checked in three levels. First, we used the positive control provide with kit in which foreign PhiX174(HaeⅢ) was added into skeletal muscle mRNA. The positive control was performed in parallel with our samples. Th is would tell us the working condition of system. Second, housekeeping gene G3PDH were amplifi ed with PCR to check our samples' subtractive efficiency. At last, 288 clones were picked up from each library and sequenced. Repeated fragments were checked using software Assembly LIGNTM. Results Every step in this experiment accord with theoretic require ments of SSH. Two subtractive libraries have been successfully set up, one contains stroke specific gene fragment, the other contains normal specific gene fragment. The volume of these two libraries was estimated t o be 7.1×104 in stroke and 5.5×105 in normal. Subtractive efficiency tests indicate that the libraries are in good quality. Conclusion Two subtractive libraries have been successfully set up, one contains stroke specific gene fragment, the other contains normal specifi c gene fragment. These two libraries have set up the fundamental for cloning of stroke related genes in our rat model.