The Microarray and Molecular Biology Module, housed in 741 square feet of space on the 5th and 2nd floors ofthe Kellogg Eye Center, facilitates vision research by providing a wide array of services and resources in theareas of microarray technology and molecular biology to the University's vision researchers and theircollaborators within and outside the institution. This Module originated from the recent merger of the MolecularBiology Module and Genetics and DNA Microarray Module, resulting in improved efficiency and the addition ofresources, techniques and services that were not previously offered by either Module. The Department ofOphthalmology and Visual Sciences plans to provide an additional 443 square feet of dedicated space for thisModule 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, fromexperimental design, RNA isolation, and probe generation, to data analysis and confirmation of results byquantitative PCR. Staffing this Module is Matthew Brooks, a full-time research associate with extensiveexperience in both molecular biology and microarray methods. Mr. Brooks assists investigators with RNAisolation techniques, including from samples excised by microdissection via the Module's Arcturus AutoPixLaser Capture Microdissection system. The use of Affymetrix GeneChips by our vision scientists for geneprofiling research is facilitated by the availability of an Affymetrix GeneChips Fluidic Station 450, an AffymetrixGeneChip Hybridization Oven 640, an Affymetrix GeneChip Scanner 3000, and an Affymetrix 428 ArrayScanner. Mr. Brooks assists investigators with probe generation, GeneChip hybridization, data acquisition, anddata analysis. In addition to the standard Affymetrix protocol, more recent techniques allowing probegeneration using total RNA amounts as low as 5 ng are also available. These non-traditional techniques ofprobe generation have proved invaluable to vision researchers here at the University of Michigan and theircollaborators in generating gene expression data from limited starting material, such as isolatedphotoreceptors and Muller cells.In addition to Affymetrix arrays, the MMB Module has considerable expertise in generation of custommicroarrays of cDNA clones or amplified promoter regions. Such custom arrays offer unique avenues ofinvestigation, 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 ashuman RPE genes for various investigators and for different projects. Efforts are underway to generate arraysof amplified DNA fragments corresponding to the promoter regions of 'eye' genes, which have significantpromise for ChIP (chromatin immunoprecipitation)-on-Chip studies that seek to identify combinatorial generegulatory networks. With such capabilities, options are endless for generating custom arrays that aredeveloped to investigator specifications.All the basic microarray analysis methods, including normalization of data, generation of lists of present/absentgenes, and lists of differentially expressed genes, are performed in-house by Ritu Khanna, M.S., a computerscientist and software engineer by training who is available on a part-time basis. More advanced statisticalanalysis is available through collaboration with Dr. Swaroop and Dr. Alfred Hero from the ElectricalEngineering and Computer Science Department. This team has developed statistical methods for analysis andclustering of microarray data, including FDR-CI, Pareto, and network constrained clustering, which are beingused 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 equipmentthat would be impractical for a single laboratory to acquire or maintain. Mr. Brooks is responsible formaintaining all of the Module's equipment and training research personnel to ensure proper use. The availableequipment 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 GeneChipHybridization Oven 640, a Kodak X-OMAT 1000A film developer, two Kodak 440CF Image Stations, two USFilter Elga Purelab Plus water purification systems, a Beckman J2-21M centrifuge, a Beckman L8-70M ultracentrifuge, 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 MolecularDevices SpectraMax 190 microplate reader, a Beckman DU530 spectrophotometer, a Perkin-Elmer LambdaBio20 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 Veritasmicroplate luminometer, an Amaxa Nucleofactor II Transfection System, and a Virtek SDDC-2 roboticmicroarrayer.Finally, the MMB Module facilitates the research of vision scientists by providing a variety of services includinggene cloning consultation, primer design, DMA modification protocols, sequence analysis, and databaseservices. These protocols have assisted participating investigators in the construction of cDNA libraries, geneexpression cassettes, and bait/prey vectors for yeast two-hybrid assays. Recombinant protein expressionusing the Baculovirus Expression System (BBS) is also available, allowing the production of large quantities ofpost-translationally modified recombinant protein. The expertise to produce and purify His-Tag and GSTfusion proteins also exists in-house. These services can advance functional studies, including thedevelopment of protein activity assays, crystal structure analysis, protein modification analysis, and antibodydevelopment.
Showing the most recent 10 out of 214 publications
