激光捕获显微切割和基因芯片技术用于肿瘤实质细胞转录组的构建

Construction of transcriptome profiling of cancerous parenchyma cells using laser capture microdissection and Gene-Chip technology

目的：联合应用激光捕获显微切割和基因芯片技术构建纯化肿瘤实质细胞转录组。方法：应用激光捕获显微切割技术从肿瘤组织冰冻切片中获取纯化肿瘤实质细胞，提取RNA并对其进行线性扩增得到aRNA，合成cRNA探针后与全基因组基因芯片Affymetrix HG—U133．Plus 2．0 Gene—Chip杂交构建全基因组表达谱。结果：从5例结肠癌组织样本中各获取了3mm。细胞，抽提获得RNA118．4～300．3ng，混合后取100ng进行线性扩增获得aRNA 7ug。基因芯片各项质控标准符合基因表达谱芯片操作指南，共有18205条转录本表达，代表15276个基因。结论：联合应用激光捕获显微切割和全基因组基因芯片技术可构建纯化肿瘤实质细胞转录组。

Objective ： To construct transcriptome profiling of parenchyma cell using laser capture microdissection （LCM） com- bined with Gene-Chip technology. Methods： We obtained cancerous parenchyma cells from frozen sections of tumor tissues by using LCM. RNA was extracted from the cells, aRNA was obtained after linear amplification, and then converted to cRNA probe, finally hy- bridized with Affymetrix HG-U133. Plus 2.0 Gene-Chip for construction of full genome transcriptome profiling. Results： About 3 mm^2 cancerous colon cells were obtained from each of 5 colon cancer specimens, respectively. The quantity of total RNA extracted from can- cerous colon cells ranged from 118.4 to 300.3 ng. We obtained 7 ug of aRNA after linear amplification from 100 ng mixed RNA. The quality control was guided by the operating instruction standard of Gene-Chip. Totally 18 205 transcripts were presented representing 15 276 genes. Conclustion： The combination of laser capture microdissection and Gene-Chip technology could be used for construction of transcriptome profiling of purified cancerous parenchyma cells.