MICROARRAY AND MOLECULAR BIOLOGY MODULE

微阵列和分子生物学模块

• 批准号: 7286539
• 负责人: ANAND SWAROOP
• 金额: $ 8.86万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7286539
• 关键词: Antibodies; Appendix; Area; Baculovirus Expression System; Biological Assay; Chimeric Proteins; Cluster Analysis; Collaborations; Computers; Consultations; Custom; DNA; DNA Microarray Chip; DNA Microarray format; Data; Data Analyses; Databases; Development; Devices; Electrical Engineering; Ensure; Equipment; Experimental Designs; Eye; Film; Floor; Gamma counter; Gene Expression; Generations; Genes; Genetic; Glutathione S-Transferase; Housing; Human; Image; Institution; Investigation; Laboratories; Methods; Michigan; Microarray Analysis; Microdissection; Modification; Molecular; Molecular Biology; Muller' s cell; Mus; Ophthalmology; Pathway interactions; Photoreceptors; Polymerase Chain Reaction; Post-Translational Protein Processing; Postdoctoral Fellow; Production; Promoter Regions; Proteins; Protocols documentation; RNA; Range; Reader; Recombinant Proteins; Regulator Genes; Research; Research Personnel; Resources; Retinal; Robotics; Sampling; Scientist; Scintillation Counter; Sequence Analysis; Services; Software Engineering; Standards of Weights and Measures; Statistical Methods; Structure; System; Techniques; Time; Training; Transfection; Two-Hybrid System Techniques; Universities; Vision; Vision research; Water Purification; Yeasts; Zebrafish; base; cDNA Arrays; cDNA Library Construction; chromatin immunoprecipitation; combinatorial; computer science; data acquisition; design; experience; eye center; foot; gene cloning; improved; laser capture microdissection; molecular dynamics; protein expression; vector; vision science; Antibodies; Appendix; Area; Baculovirus Expression System; Biological Assay; Chimeric Proteins; Cluster Analysis; Collaborations; Computers; Consultations; Custom; DNA; DNA Microarray Chip; DNA Microarray format; Data; Data Analyses; Databases; Development; Devices; Electrical Engineering; Ensure; Equipment; Experimental Designs; Eye; Film; Floor; Gamma counter; Gene Expression; Generations; Genes; Genetic; Glutathione S-Transferase; Housing; Human; Image; Institution; Investigation; Laboratories; Methods; Michigan; Microarray Analysis; Microdissection; Modification; Molecular; Molecular Biology; Muller' s cell; Mus; Ophthalmology; Pathway interactions; Photoreceptors; Polymerase Chain Reaction; Post-Translational Protein Processing; Postdoctoral Fellow; Production; Promoter Regions; Proteins; Protocols documentation; RNA; Range; Reader; Recombinant Proteins; Regulator Genes; Research; Research Personnel; Resources; Retinal; Robotics; Sampling; Scientist; Scintillation Counter; Sequence Analysis; Services; Software Engineering; Standards of Weights and Measures; Statistical Methods; Structure; System; Techniques; Time; Training; Transfection; Two-Hybrid System Techniques; Universities; Vision; Vision research; Water Purification; Yeasts; Zebrafish; base; cDNA Arrays; cDNA Library Construction; chromatin immunoprecipitation; combinatorial; computer science; data acquisition; design; experience; eye center; foot; gene cloning; improved; laser capture microdissection; molecular dynamics; protein expression; vector; vision science

The Microarray and Molecular Biology Module, housed in 741 square feet of space on the 5th and 2nd floors of the Kellogg Eye Center, facilitates vision research by providing a wide array of services and resources in the areas of microarray technology and molecular biology to the University's vision researchers and their collaborators within and outside the institution. This Module originated from the recent merger of the Molecular Biology Module and Genetics and DNA Microarray Module, resulting in improved efficiency and the addition of resources, techniques and services that were not previously offered by either Module. The Department of Ophthalmology and Visual Sciences plans to provide an additional 443 square feet of dedicated space for this Module on the 7th floor of the new building in early 2010 (see Appendix C). The MMB Module provides a comprehensive range of services required for microarray research, from experimental design, RNA isolation, and probe generation, to data analysis and confirmation of results by quantitative PCR. Staffing this Module is Matthew Brooks, a full-time research associate with extensive experience in both molecular biology and microarray methods. Mr. Brooks assists investigators with RNA isolation techniques, including from samples excised by microdissection via the Module's Arcturus AutoPix Laser Capture Microdissection system. The use of Affymetrix GeneChips by our vision scientists for gene profiling research is facilitated by the availability of an Affymetrix GeneChips Fluidic Station 450, an Affymetrix GeneChip Hybridization Oven 640, an Affymetrix GeneChip Scanner 3000, and an Affymetrix 428 Array Scanner. Mr. Brooks assists investigators with probe generation, GeneChip hybridization, data acquisition, and data analysis. In addition to the standard Affymetrix protocol, more recent techniques allowing probe generation using total RNA amounts as low as 5 ng are also available. These non-traditional techniques of probe generation have proved invaluable to vision researchers here at the University of Michigan and their collaborators in generating gene expression data from limited starting material, such as isolated photoreceptors and Muller cells. In addition to Affymetrix arrays, the MMB Module has considerable expertise in generation of custom microarrays of cDNA clones or amplified promoter regions. Such custom arrays offer unique avenues of investigation, i.e., a focus on a specific set of functional genes or those not present on Affymetrix GeneChips. The Module has produced custom cDNA arrays of human, mouse and zebrafish retinal genes as well as human RPE genes for various investigators and for different projects. Efforts are underway to generate arrays of amplified DNA fragments corresponding to the promoter regions of "eye" genes, which have significant promise for ChIP (chromatin immunoprecipitation)-on-Chip studies that seek to identify combinatorial gene regulatory networks. With such capabilities, options are endless for generating custom arrays that are developed to investigator specifications. All the basic microarray analysis methods, including normalization of data, generation of lists of present/absent genes, and lists of differentially expressed genes, are performed in-house by Ritu Khanna, M.S., a computer scientist and software engineer by training who is available on a part-time basis. More advanced statistical analysis is available through collaboration with Dr. Swaroop and Dr. Alfred Hero from the Electrical Engineering and Computer Science Department. This team has developed statistical methods for analysis and clustering of microarray data, including FDR-CI, Pareto, and network constrained clustering, which are being used extensively. New methods are being pursued to construct gene regulatory networks and pathways. Another major function of the MMB Module is to provide access to and training for small and large equipment that would be impractical for a single laboratory to acquire or maintain. Mr. Brooks is responsible for maintaining all of the Module's equipment and training research personnel to ensure proper use. The available equipment include: a Molecular Dynamics Storm 840 Phosphoimager, an Affymetrix GeneChip Scanner 3000, an Affymetrix 428 Array Scanner, an Affymetrix GeneChips Fluidic Station 450, an Affymetrix GeneChip Hybridization Oven 640, a Kodak X-OMAT 1000A film developer, two Kodak 440CF Image Stations, two US Filter Elga Purelab Plus water purification systems, a Beckman J2-21M centrifuge, a Beckman L8-70M ultra centrifuge, a Beckman Optima L-90K ultra centrifuge, 2 Integrated SpeedVac ISS110 systems, a Tuttnauer- Brinckman 3870E tabletop autoclave, an Arcturus AutoPix Laser Capture Microdissection system, a Molecular Devices SpectraMax 190 microplate reader, a Beckman DU530 spectrophotometer, a Perkin-Elmer Lambda Bio20 spectrophotometer, a Thermo Spectronic French Press, a Beckman 5500 gamma counter, a Perkin- Elmer WinSpectral 1414 scintillation counter, an Alpha Innotech Image Station, a Turner BioSystems Veritas microplate luminometer, an Amaxa Nucleofactor II Transfection System, and a Virtek SDDC-2 robotic microarrayer. Finally, the MMB Module facilitates the research of vision scientists by providing a variety of services including gene cloning consultation, primer design, DMA modification protocols, sequence analysis, and database services. These protocols have assisted participating investigators in the construction of cDNA libraries, gene expression cassettes, and bait/prey vectors for yeast two-hybrid assays. Recombinant protein expression using the Baculovirus Expression System (BBS) is also available, allowing the production of large quantities of post-translationally modified recombinant protein. The expertise to produce and purify His-Tag and GST fusion proteins also exists in-house. These services can advance functional studies, including the development of protein activity assays, crystal structure analysis, protein modification analysis, and antibody development.

微阵列和分子生物学模块，位于741平方英尺的空间，位于5层和第二层 Kellogg眼中中心通过在 微阵列技术和分子生物学领域，大学的视野研究人员及 机构内外的合作者。该模块起源于分子的最近合并 生物学模块和遗传学和DNA微阵列模块，导致效率提高，并增加 以前没有任何模块提供的资源，技术和服务。部门 眼科和视觉科学计划为此提供另外443平方英尺的专用空间 2010年初的新建筑物7楼的模块（请参阅附录C）。 MMB模块提供了微阵列研究所需的全面服务， 实验设计，RNA隔离和探测生成，以通过 定量PCR。配备此模块的是Matthew Brooks，他是一位全日制研究助理 具有分子生物学和微阵列方法的经验。布鲁克斯先生协助调查人员进行RNA 隔离技术，包括通过微分辨率通过模块的Arcturus Autopix切除的样品 激光捕获微分解系统。我们的视觉科学家使用Affymetrix Genechips用于基因 分析研究通过Affymetrix Genechips Fluidic Station 450（Affymetrix）的可用性促进了研究 Genechip杂交烤箱640，Affymetrix Genechip Scanner 3000和Affymetrix 428阵列 扫描器。布鲁克斯先生协助研究人员进行探测，杂交，数据获取和 数据分析。除了标准Affymetrix协议外，最新技术允许探针 使用总RNA量低至5 ng的生成。这些非传统技术 事实证明，探测器对密歇根大学的视力研究人员及其 从有限的起始材料中生成基因表达数据的合作者，例如孤立的 感光体和穆勒细胞。 除Affymetrix阵列外，MMB模块在生成自定义方面还具有丰富的专业知识 cDNA克隆或放大启动子区域的微阵列。这样的自定义阵列提供了独特的途径 研究，即关注一组功能基因或不存在于Affymetrix Genechips的特定功能基因。 该模块产生了人，小鼠和斑马鱼视网膜基因的定制cDNA阵列以及 针对各种研究人员和不同项目的人类RPE基因。正在努力生成阵列 与“眼”基因的启动子区域相对应的放大DNA片段 芯片的诺言（染色质免疫沉淀） - 芯片研究，试图鉴定组合基因 监管网络。具有这样的功能，选择是无限的，用于生成自定义数组 开发到研究者规格。 所有基本的微阵列分析方法，包括数据的归一化，当前/缺失列表的生成 基因和差异表达基因的列表由计算机的Ritu Khanna在内部执行 培训的科学家和软件工程师可以兼职。更先进的统计 可以通过与电气的Swaroop博士和Alfred Hero博士合作获得分析 工程和计算机科学系。该团队开发了分析的统计方法和 微阵列数据的聚类，包括FDR-CI，Pareto和网络约束聚类，正在 广泛使用。正在追求新的方法来构建基因调节网络和途径。 MMB模块的另一个主要功能是为小型和大型设备提供访问和培训 对于一个实验室而言，这是不切实际的。布鲁克斯先生负责 维护所有模块的设备和培训研究人员，以确保正确使用。可用 设备包括：分子动力学风暴840 Phosphoimager，Affymetrix Genechip Scanner 3000， Affymetrix 428阵列扫描仪，Affymetrix Genechips流体站450，Affymetrix Genechip 杂交烤箱640，一个柯达X-Amat 1000A膜开发人员，两个柯达440cf图像站，两个美国 过滤器ELGA PURELAB加水净化系统，Beckman J2-21M离心机，Beckman L8-70m Ultra 离心机，Beckman Optima L-90K Ultra离心机，2集成SpeedVac ISS110系统，一个Tuttnauer- Brinckman 3870E桌面高压灭菌器，Arcturus Autopix激光捕获微解剖系统，一种分子 设备Spectramax 190微板读取器，Beckman DU530分光光度计，Perkin-Elmer Lambda Bio20分光光度计，热光谱法国出版社，贝克曼5500伽玛计数器，perkin- Elmer Winspectral 1414闪烁柜台，Alpha Innotech Image Station，Turner Biosystems Veritas 微板光发光仪，Amaxa核素II转染系统和Viverek SDDC-2机器人 微阵列。 最后，MMB模块通过提供各种服务，包括 基因克隆咨询，底漆设计，DMA修改协议，序列分析和数据库 服务。这些方案已协助参与研究人员构建cDNA库，基因 酵母两杂化测定法的表达盒和诱饵/猎物矢量。重组蛋白表达 还可以使用杆状病毒表达系统（BBS），可以产生大量 翻译后修饰的重组蛋白。生产和净化His-Tag和GST的专业知识 融合蛋白也内部也存在。这些服务可以推进功能研究，包括 蛋白质活性测定，晶体结构分析，蛋白质修饰分析和抗体的开发 发展。