Espina Virginia, Wulfkuhle Julia D, Calvert Valerie S, VanMeter Amy, Zhou Weidong, Coukos George, Geho David H, Petricoin Emanuel F, Liotta Lance A
Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Blvd. MS 4E3, Manassas, Virginia, USA.
Nat Protoc. 2006;1(2):586-603. doi: 10.1038/nprot.2006.85.
Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1-1.5 h.
解析在组织微环境中驱动疾病的细胞和分子相互作用，有望发现未来的药物靶点。为了通过分子分析重现体内相互作用，必须能够在异质组织微生态环境中分析特定细胞群体。激光捕获显微切割（LCM）是一种在直接显微镜观察下获取组织细胞亚群的方法。LCM技术可以直接收获感兴趣的细胞，也可以通过切除不需要的细胞来分离特定细胞，从而得到组织学上纯净的富集细胞群体。存在多种下游应用：DNA基因分型和杂合性缺失（LOH）分析、RNA转录谱分析、cDNA文库构建、蛋白质组学发现和信号通路分析。在此，我们对LCM技术进行全面描述，重点是来自LCM用户的提示和故障排除建议。执行此方案所需的总时间通常为1-1.5小时。
