Cho-Vega Jeong Hee, Troncoso Patricia, Do Kim-Anh, Rago Carlo, Wang Xuemei, Tsavachidis Spiridon, Medeiros L Jeffrey, Spurgers Kevin, Logothetis Christopher, McDonnell Timothy J
Department of Molecular Pathology, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA.
Mod Pathol. 2005 Apr;18(4):577-84. doi: 10.1038/modpathol.3800327.
Cell-specific gene expression profiling from heterogeneous human tissues is confounded by cell purification limitations. Here, we describe a technique to generate gene expression profiles of pure populations of prostate cancer cells obtained from fresh-frozen prostatectomy specimens and small initial quantities of RNA by combining laser capture microdissection and microserial analysis of gene expression (LCM-microSAGE). Two microSAGE libraries were obtained from approximately 100,000 laser pulses, estimated to contain fewer than 3 x 10(5) cells and 20-30 ng mRNA. Two libraries were sequenced to a depth of 10,111 and 10,463 unique tags from normal and cancer cells, representing 6453 and 6923 genes, respectively. Most transcripts were expressed at similar levels, but cancer cells compared with normal cells had increased expression of 385 tags and decreased expression of 389 tags. A total of 20 genes were differentially expressed (P<0.05); five of these genes were upregulated and 15 were downregulated in cancer cells. Quantitative reverse transcriptase-polymerase chain reaction results from three selected genes corroborated the existence of cell-specific gene expression in LCM-microSAGE-derived libraries. In conclusion, the LCM-microSAGE approach demonstrates that large-scale expression profiles of known and unknown transcripts can be generated from pure populations of target cells obtained from human tissue samples comprised of heterogeneous mixtures of cell types.
从异质性人类组织中进行细胞特异性基因表达谱分析受到细胞纯化局限性的困扰。在此，我们描述了一种技术，通过结合激光捕获显微切割和基因表达微序列分析（LCM-微SAGE），从新鲜冷冻前列腺切除标本和少量初始RNA中获得前列腺癌细胞纯群体的基因表达谱。从大约100,000次激光脉冲中获得了两个微SAGE文库，估计包含少于3×10⁵个细胞和20 - 30 ng mRNA。对两个文库进行测序，来自正常细胞和癌细胞的独特标签深度分别为10,111和10,463，分别代表6453和6923个基因。大多数转录本以相似水平表达，但与正常细胞相比，癌细胞中385个标签表达增加，389个标签表达减少。共有20个基因差异表达（P<0.05）；其中5个基因在癌细胞中上调，15个基因下调。对三个选定基因的定量逆转录聚合酶链反应结果证实了LCM-微SAGE衍生文库中存在细胞特异性基因表达。总之，LCM-微SAGE方法表明，可以从由细胞类型异质混合物组成的人类组织样本中获得的靶细胞纯群体中生成已知和未知转录本的大规模表达谱。
