Massively Parallel Gene Expression Analysis

本申请中提出的研究将开发和利用大规模并行基因分析技术。既将使用Affymetrix公司的商用微阵列，也将使用该工厂定制的阵列。这些实验的基本策略是确定与个别实验项目相关的基因。这些有限的基因集将被包括在我们设施制造的阵列中，这些阵列将为个人用户定制。这种方法的优点是，多个实验将以成本效益高的方式进行，允许对数据进行适当的统计分析，以及系统的变化和操纵。正在进行的研究是由NIDDK资助的研究人员在人类、动物和同种细胞中检测疾病过程和模型的研究：1)在实验性糖尿病过程中发生的小鼠肾病；2)实验性炎症性肠病(IBD)小鼠的淋巴细胞异常；3)肠上皮细胞对腔钠负荷变化的适应的分子基础；4)IBD患者单核细胞激活缺陷；5)类固醇对多囊卵巢综合征肌肉和脂肪细胞基因表达的影响；6)甲状腺激素抵抗小鼠模型中信号缺陷的分析；7)甲状腺激素抵抗患者信号缺陷的分析；8)为移植准备的人胰岛的mRNA变化分析；9)高糖暴露对胰岛β细胞的影响；以及10)促性腺激素释放激素对神经元基因表达的响应。这些分析将使识别在这些不同条件下上调或下调的相关基因成为可能。试点和可行性研究将是：1)开发来自垂体、胰岛和肾小球的基因微阵列，以便从这些解剖独特的位置获得基因图谱；以及2)利用激光捕获显微解剖显微镜从狼疮性肾炎的动物模型和人类组织标本中获取肾小球RNA，并随后应用微阵列技术来识别这种疾病的基因变化。如果这个由NIDDK赞助的核心设施获得资助，将开展研究各种肾脏、内分泌和胃肠道疾病模型和过程中基因表达的创新工作。

The research proposed in this application will develop and utilize massively parallel gene analysis technology. Both commercially available microarrays from Affymetrix and arrays custom-made in this facility will be used. The basic strategy for these experiments is to identify genes that are relevant for individual experimental projects. These finite gene sets will be included on arrays made in our facility, which will be customized for the individual user. The advantages of such an approach are that multiple experiments will then be performed cost effectively, allowing appropriate statistical analyses of data, as well as systematic variations and manipulations. Ongoing studies are those from NIDDK-funded investigators examining disease processes and models in humans, animals and homogenous cells: 1) Nephropathy occurring during the course of experimental diabetes mellitus in mice; 2) Lymphocytic abnormalities in mice with experimental inflammatory bowel disease (IBD); 3) Molecular basis of intestinal epithelial adaptation to altered luminal sodium load; 4) Activation defects in monocytes from patients with IBD; 5) Effects of steroids on muscle and adipocyte gene expression in polycystic ovary syndrome; 6) Analysis of signaling defects in mouse models of thyroid hormone resistance; 7) Analysis of signaling defects in patients with thyroid hormone resistance; 8) Analyses of mRNA changes in human pancreatic islets prepared for transplantation; 9) Effects of high glucose exposure to pancreatic beta cells; and, 10) Neuronal gene expression in response to gonadotropin-releasing hormone. These analyses will permit the identification of relevant genes that are upregulated or downregulated in these various conditions. Pilot and feasibility studies will be to: 1) Develop cDNA microarrays from pituitary, pancreatic islets and renal glomeruli to allow gene profiling from these anatomically unique sites; and 2) Utilize laser capture microdissection microscopy to obtain glomerular RNA from animal models and human tissue specimens of lupus nephritis, and subsequently apply microarray technology to identify gene changes in this disease. If this NIDDK-sponsored core facility is funded, innovative work investigating gene expression in a variety of renal, endocrinologic and gastrointestinal disease models and processes will be performed.