The Core Genomics Facility By Carol Westbrook, MD, Ph.D. Overview The Research Resources Center’s Core Genomics Facility (CGF) is the newest of the laboratories and services that provide technical support to the research faculty at the University of Illinois at Chicago (UIC). The mission of the CGF is to provide state-of-the art equipment and technical support for genomics research, particularly microarrays. The facility is now providing services including, hybridization and scanning of commercial slides Affymetrix array hybridization, and the custom fabrication of small glass slide arrays, as well as bioinformatics support for these activities. The facility also houses the 40,000 sequence-verified human cDNA UniGene clones, and has licensed software specifically for analysis of arrays, including ScanArray, QuantArray, GeneSpring, Spotfire, and GeneChip. The CGF contains several sophisticated robotics systems, including the Total Array System from Biorobotics for cDNA array production, the BioRobot 8000 liquid handler from Qiagen, and an MWG Biotech multiblock system for high-throughput PCR. Also housed in the facility are the GeneChip system from Affymetrix, and a dual-laser confocal scanner ScanArray Lite (Packard BioChip Technologies). The scanner is available for all UIC investigators, including those who purchased commercial arrays and hybridized them in their own laboratories but need to scan and analyze them. The equipment, staff, and services of the CGF are described in detail on the CGF services web page: http://www.rrc.uic.edu/SERVICES/CGF/. The site also contains useful microarray-related links, such as a partial list of commercial microarray providers, and academic pricing for Affymetrix arrays. History of the Core Genomics Facility The concept for the CGF was developed by the “Core Genomics Facility Task Force” which convened in May, 1999 after a campus-wide meeting to judge faculty interest in developing a microarray facility at UIC. The Task Force, chaired by Dr. Lon Kaufman, met over a five month period to evaluate available equipment and systems, and presented its report to the Provost in February, 2000, outlining the staffing and equipment needs for a core facility managed by the RRC. An initial equipment budget of $285,000 was recommended, and the funds were contributed equally by the Office of the Vice Chancellor for Research (OVCR), the RRC and the provost. Dr. Carol Westbrook agreed to head the facility and equipment bids went out with most purchases completed by May, 2000. By August of 2000, with the staff appointments of Dr. Zarema Arbieva and Mr. Seby Edassery, the facility was in place.	CGF Staff Pictured (left to right): Zarema Arbieva, Seby Edassery, Peter Larsen, and Carol Westbrook. Dr. Carol Westbrook, MD, Ph.D. (Departments of Medicine, Molecular Genetics, and Bioengineering) is the faculty Director of the RRC Core Genomics Facility. Dr. Westbrook has had a life-long interest in genomics, which arose out of her research in cancer genetics and chromosomal translocations. Dr. Zarema Arbieva, Ph.D. (Department of Medicine and Research Resources Center) is assistant director of the facility. She has spent five years working in the area of gene discovery in breast and colon cancers and has developed a deep interest in applying modern genomics technologies and strategies to cancer genetics. She oversees all the facility activities and provides specialized consultations in experimental design. Seby Edassery, MS (Research Resources Center) is a research programmer. He has masters’ degrees in biochemistry and biotechnology and experience in programming and database management. Seby assists customers with bioinformatics tasks, and maintains the genomics databases. Peter Larsen (Research Resources Center) is a Research Specialist who joined the CGF staff in June, 2001. He has worked with many Chicago area biotech companies, and most recently with Vysis (Downers Grove, Illinois) developing production methodologies, quality control protocols, and hybridization techniques for genomic microarrays. Peter performs Affymetrix and glass slide microarray hybridizations, and is also involved in printing custom glass microarrays.
Unfortunately, the Task Force had overlooked an important consideration: there was no request for space to house the CGF. In order to come online as quickly as possible and to begin training on the equipment, Dr. Westbrook offered space in her lab at MBRB (Molecular Biology Research Building). Plans to move the facility to College of Medicine - West were abandoned due to the high cost of renovation to meet the ventilation and safety requirements. The CGF remains in MBRB, part in Dr. Westbrook’s lab and part in space belonging to the Department of Medicine. The faculty and staff at the W. M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign (UIUC), especially Mark Band and Harris Lewin, were tremendously helpful in getting the facility up and running, sharing protocols and experience. The collaboration between the UIC and UIUC groups was so successful that they joined forces to submit a proposal to the University’s Intercampus Research Initiative in Biotechnology (IRIB) program. The IRIB proposal, “Affymetrix Technology Platform for the Genomic Analysis of Women’s Neoplasms,” was successful and brought the Affymetrix equipment to both campuses. On March 16, 2001, the RRC hosted a day-long seminar, “Microarray Technology: Applications and Approaches,” which featured presentations by genomic scientists from universities and industry, and brought this technology to the forefront for the first time at UIC. This presentation was well-timed, as the completion of the human genome sequence had just been announced, and UIC scientists’ interest in genomics -- and their demand for services -- picked up considerably. By spring it was clear that additional staff would be needed in the Core Facility to manage the new equipment--a robotic liquid handler and the GeneChip system--but especially to provide for the increasing Bioinformatics needs. After an extensive search, Mr. Edassery was promoted to the position of Scientific Programmer, and Mr. Peter Larsen was hired as the staff scientific research specialist in August, 2001. The Research Resources Center Core Genomics Facility continues to grow in its interests and in its services. Recently, a joint effort between the RRC and the Department of Ophthalmology, headed by Dr. Beatrice Yue, brought in grant funds from the National Eye Institute to improve genomic facilities. As a result, the RRC will be able to purchase additional equipment and cDNA libraries but, most importantly, it will expand its bioinformatics capabilities. To the extent that technology and computational biology represent the future of biological research, the CGF is committed to its development at UIC. Available Services (Photo at left) Affymetrix chip being placed into the scanner. Affymetrix arrays: The Core Genomics Facility is currently offering hybridization and scanning of Affymetrix GeneChips using the GeneChip system. GeneChips are miniaturized, high-density arrays of thousands or tens-of thousands of oligonucleotide probes (16-mers) which are synthesized directly on the slides (See Figure). The oligos represent known genes and ESTs. Affymetrix makes GeneChips for multiple organisms including Human, Mouse, Rat, and Yeast. Detailed information on the spectrum of arrays and their cost is available at http://www.rrc.uic.edu/SERVICES/CGF/Affymetrix_price_list.html. See the box for details of Affymetrix studies. cDNA arrays: The CGF is currently offering hybridization and scanning of commercially available cDNA arrays. A limited list of array suppliers is presented on the web page to assist customers to find the most suitable array for their experiments. Glass slide arrays contain PCR-amplified fragments of cDNA clones spotted on pre-treated glass slides. Unlike Affymetrix arrays, glass slide arrays are used for differential hybridization experiments. RNA from experimental and control samples are each labeled with a different fluorescent dye (Cy3 and Cy5) and hybridized to a single array in a competitive hybridization reaction. The signal from each RNA that is hybridized to the array is quantitated by laser excitation of the individual fluorescent markers using a confocal scanning laser microscope. The Cy3 to Cy5 ratio is measured, providing an estimate of the relative intensity of the reference and test samples for each cDNA on the array. Custom array printing: The facility currently prints small custom arrays containing up to 1000 cDNA clones. The user can provide his or her own clones, or select them from the IMAGE cDNA clone collection. Printing an array requires considerable preparation. First, the inserts from cDNA clones are PCR-amplified using well-defined vector-based primers flanking the cDNA inserts. The amplified products are then column-purified, run on a gel for quality control and quantified and re-arrayed into 96- or 384-well plates. Printing, using the robotic Total Array System, follows this process; several dozen arrays can be printed at a time. The printed arrays are then baked and chemically blocked, ready for hybridization. IMAGE cDNA collection: UIC licensed a large collection of more than 40,000 human IMAGE cDNA clones from Research Genetics, representing more than 35,000 Unigene clusters. Information on these clones is available through the online CGF database at http://westsun.hema.uic.edu/cgi-bin/db.cgi?db=ResGen which is frequently updated to reflect the most current Unigene release. Clones are stored as glycerol stocks in 96-well plates and are available to all UIC investigators free of charge. The CGF will select and array custom sets of these clones for chipss or other research needs. Bioinformatics: A genomics facility requires intensive computing for its day-to-day operations, such as selection of cDNAs, high-throughput PCR, tracking slides and summarizing hybridization results from Affymetrix and glass slides. The available software packages, such as GeneSpring and Spotfire, enable users to perform clustering and data mining with the assistance of the RRC staff. A well-planned series of microarray experiments can be executed in a few days, but the computational studies can take months. The CGF offers a variety of bioinformatics services and assistance starting from the initial steps of the custom array design or input data organization. Personnel will assist customers with: Custom selection of cDNA clones from the IMAGE collection. Array scanning and data acquisition using ScanArray and QuantArray software packages, followed by preliminary identification of differentially-expressed genes relative to experimental variables. Tables of candidates to be evaluated by other methods or analyzed statistically or biologically are produced at this step. Cluster analysis to detect groups of concordantly-expressed genes. Two software packages (Spotfire and Genespring) are available for this analysis. Researchers can access many publicly available expression and annotation databases, thus creating a critical link between sets of individual experimental results and a wealth of readily available biological information.
Some of Our Users Dr. Alan Diamond, who is interested in the mechanism by which the essential trace element selenium prevents cancer, and Dr. Beatrice Yue, who studies differentially-expressed genes in an ophthalmologic disease known as “keratoconus,” have made use of the bioinformatics services to analyze experimental data that they have collected with collaborators. Dr. Yogen Saunthararajah is analyzing a mouse which is transgenic for the ETO gene using glass slide microarrays purchased from the University of Toronto. Dr. Zarema Arbieva, as a member of the IRIB group interested in “Women’s Cancers,“ has designed a breast-cancer chip, containing approximately 4000 genes. The chip will be printed this winter, and will be used to analyze breast and endometrial cancer in collaboration with several investigators, including Dr. Rajeswari Mehta, Dr. Serdar Bulun, both at UIC, and a number of investigators at UIUC. Dr. Irwin Brodsky is conducting an exploratory study of hypoglycemia in healthy adults, using Affymetrix arrays.	How do you use Affymetrix GeneChips? High quality RNA - at least 10µg -- is essential to the process. cDNA is synthesized from the RNA by reverse transcription reaction. The cDNA is biotinylated, and the labeled material is hybridized to the GeneChip, followed by washes in the Affymetrix fluidics station in the presence of streptavidin-conjugated fluorescent marker. GeneChips are then scanned in the dedicated Affymetrix scanner in the single emission detection channel and the amount of the bound probe is calculated. Comparative analysis are performed using normalized readings off the two arrays. How do you analyze Affymetrix GeneChip data? Data is analyzed using GeneChip expression analysis software. Data is reported per clone as “present,” “absent,” or “marginal.” Compared to another chip, genes and ESTs are reported as “increased,” “decreased,” or “no change” with a range of Log Ratio with a 95% confidence interval. Additional assistance in analyzing GeneChip data can be obtained from the CGF’s bioinformatics services. Analysis of Affymetrix chips using NetAffyx Users may have access to Affymetrix’s NetAffyx (www.netaffx.com) once they obtain an access code from the CGF. NetAffyx is a resource created by Affymetrix to assist researchers with the design and analysis of DNA array-based experiments. This system provides convenient access to the information underlying the design of Affymetrix probe arrays. NetAffyx contains a broad range of searchable resources and Affymetrix’s proprietary databases.
Future Directions Bioinformatics: Most users recognize that sophisticated mathematical expertise is needed to get the most out of their data. In order to provide this service and to facilitate collaboration between biologists and computer scientists, the RRC is partnering with the Department of Bioengineering and the Department of Ophthalmology to establish a bioinformatics facility. Plans are already in place to purchase high-speed servers and computers for parallel processing. This facility, which will provide RRC service as well as academic computing under the direction of Dr. Jie Liang, will be described in a future issue of the RRC Reporter. Improved array production and specialized chips: The current ability of the CGF to print glass slide microarrays is limited by its equipment. Small arrays of 200 to 1,000 elements can be printed reliably in small lots. However, the capability to print hundreds of high-density arrays of 10,000 or more cDNAs from libraries of human and other species, as originally envisioned by the Genomics Task Force, has yet to be realized. The Research Resources Center is now considering the purchase of a robotic arrayer that has this capability.
RRC Updates ELECTRON MICROSCOPY SERVICE (EMS) Materials Science TEM/STEM The JEOL JEM-2010F will receive a significant upgrade in the first quarter of 2002 when JEOL installs their FasTem integrated control system. As users of this instrument are aware, operation is complicated by having five computer systems (with four different operating systems) controlling the microscope and its peripherals. The FasTem system will replace these separate systems with one integrated system so that the user may deal with one computer interface. In principle this interface does not even have to be in the same room as the microscope and, in fact, the system is set up so that the microscope can be operated over the internet with appropriate software. During demonstrations of this system to potential JEOL users at UIC, microscopes in Boston, Pleasanton, California and Urbana-Champaign have been operated from UIC. This upgrade will be free of charge to RRC as a result of our close working relationship with JEOL which has seen both our JEM-2010F and JEM-3010 used as demonstration instruments for the company. The JEOL JEM-3010 has seen significantly more use over the last twelve months thanks in part to the installation of a Thermo Noran X-ray detection system in Fall 2000. Chemical analysis by X-ray is now available on five out of the eight electron microscopes in the RRC. Work is progressing on getting the VG Microscopes HB601UX operational. Progress has been slower than hoped and a number of problems have been encountered because the instrument has not been in operation since December 1997. By the end of 2001 the only remaining problems were with the scanning system and objective lens power supply. Once these problems have been resolved and the microscope checked out, the existing vacuum system will be replaced by a dry pumping system using ion pumps and turbo pumps. Meanwhile the STEM aberration corrector, being purchased from Nion Corporation in Seattle, is in the final testing stage in the factory and is expected to be delivered to the University during the first quarter of 2002. We will also be replacing the existing Parallel Electron Energy Loss Spectrometer (PEELS) with a new Ultra-High Vacuum compatible Enfina 100 PEELS from Gatan. Funding for this project has come from a National Science Foundation award to Nigel Browning (Physics), Yuri Gogotsi (Mechanical Engineering), Alan Nicholls (RRC), Christos Takoudis (Chemical Engineering) and Siva Sivanathan (Physics). Life Science TEM The JEOL JEM-100CX in MSB (Medical Sciences Building), purchased in 1978, has reached the end of its useful life and will be retired. A ComEd power-cut early in 2001 seems to have resulted in significant oil contamination of the column and the current level of use of TEM by life scientists does not justify the expense of its repair. Scanning Electron Microscopy The JEOL JXA-733, which has not seen much use since it was installed in RRC-E in 1998, looks to have a much busier year in front of it. EMS has entered an agreement with the Planetary Studies Foundation (PSF) to allow the foundation access to the JXA-733 for a minimum of 100 days during 2002. This will allow the PSF to analyse meteorites from the James M. DuPont Meteorite Collection, which they have received from the DuPont family. In the MSB laboratory the Hitachi S-3000N, installed in August 2000, has now settled in as our main research SEM. Its ease of use and the ability to look at all dry samples without modification by coating, whether insulating or not, have made it popular with many users. However, it is not the only SEM available in MSB - the JEOL JSM-6320F is still one of the highest resolution SEMs available today with a resolution of 1.2nm at 15kV and 2.5nm at 1kV. Many non-conducting specimens can be imaged in the JSM-6320F at low accelerating voltages without significant charging. The microscope is equipped with two secondary detectors (one in lens for high resolution imaging and one below the lens for topographical imaging), a backscatter detector and an X-ray detector. BIOSTATISTICS SERVICE (BSS) Minu K. Patel has accepted an invitation to be a speaker at the 12th international meeting of the World Organization of Systems and Cybernetics, scheduled for March 24 to 26 in Pittsburgh. Minu will address the meeting at a “Symposium on Creative Development.” Minu announced in the RRC staff meeting in January that he is retiring from full-time service to the university at the end of May, 2002. At the time of his retirement Minu will have served the University of Illinois for 35 years, as a provider of statistical services to UIC research investigators and students in the Research Resources Center, where he was director of the Biostatistics Service. Minu was originally hired by Dr. John P. Marbarger, the first director of the RRC. The College of Nursing, Department of Medical Surgical Nursing, has requested that he return to work part-time after his retirement, continuing to teach courses in statistics as he has done in that department for many years. His colleagues at the RRC (and we are sure, many campus investigators) will miss Minu, and we wish him the absolute happiest of “retirements.”

Wed Feb 13 19:07:39 CST 2002