摘要
目的为筛选食管癌相关的差异表达基因和基因组,建立哈萨克族食管癌表达文库。方法应用DAzLE系统构建哈萨克族食管癌cDNA表达文库,包括Trizol一步法提取总RNA,Oligo(dT)纤维素层析柱纯化mRNA,Super Script TMChoice System For cDNA Synthesis Kit合成cDNA,分级分离,纯化cDNA与质粒载体pSPORT1连接,电转化导入E.coliDH10B。结果14-18回收管合并,得到1.3×10^6个克隆,同时合成了肿瘤组和对照组的cDNA探针。结论成功构建的哈萨克族食管癌cDNA表达文库,可用于进一步筛选食管癌相关的差异表达基因,而且为搭建差异表达技术平台做好了准备工作。
Objective To establish cDNA expression library of Kazak′s esophageal cancer in Xinjiang for screening the differential expression gene and genome.Methods A cDNA library of Kazak′s esophageal cancer was constructed with the method of DAzLE.Total RNA was isolated by the Trizol reagent using single-step method.Poly(A)-RNA was purified from total RNA by using an oligo(dT) cellulose column.cDNA was synthesized using SuperScriptTM Choice System For cDNA Synthesis Kit.After cDNA size fractionation was purified by chromatography column,the double cDNA was ligated to pSPORT1 plasmid vector.Vector-ligated cDNAs were introduced into DH10B component cells by electroporation.Results Selected tubes 14 through 18,the directional library confirmed to be about 1.3×10^6 independent clones.At the same time,appropriate cDNA probes from cancer and normal tissue were specifically synthesized.Conclusions The cDNA expression library of Kazak′s esophageal cancer is successfully constructed to meet the currently recognized standards,and can be well applicable in screening cDNA-cloned genes of Kazak′s esophageal cancer.Furthermore,these results have provided further preparing for differential expression display.
出处
《地方病通报》
2008年第4期10-13,共4页
Endemic Diseases Bulletin
基金
新疆维吾尔自治区科技厅重点实验室开放课题(XJDX0208-2005-01)