The Aims of the MicroArray Core are to:
- provide high-quality services for microarray-based gene expression analysis for UofL HSC users
- educate and train users in applications, strengths, and limitations of microarray technology
- assist users in experimental design, sample processing, and data analysis
- maintain close intellectual and logistic interactions with the Dept. of Bioinformatics & Biostatistics to support customer needs in experimental design, data analysis and interpretation
- provide logistic support for grant applications and manuscripts applying microarray technology
These Aims are accomplished by providing the following services and resources to accommodate the needs of both users with prior expertise as well as untrained and inexperienced novice users:
- Sample preparation for microarray analysis
- RNA amplifications from small-size samples
- Quality and quantity assessment of samples
- Array hybridizations and primary data collections
- Assistance in comparative data analysis with Affymetrix or 3rd party software
- Assistance in more complex data-mining, statistical evaluations, pathway analysis
- DNA and RNA analysis with Agilent Bioanalyzer
- SNP analysis on human or mouse genomic DNAs
- Individual training of users on technology, molecular procedures, and primary data analysis
- Organization of training seminars, workshops, and web-seminars offered by corporate providers
Organization of the Facility
The Facility is staffed with an experienced Core Director and a full-time Research Technologist responsible for the daily operations and management, including an Associate Director. An Advisory Committee, composed of several UofL Health Science Center faculty with experience and knowledge related to microarray technology and tissue analysis, are available for consultation, oversight, and decision-making processes. One important function of the Core is the continuing state-of-the art training of current and future users by hands-on wetlab and software training sessions, combined with regular classroom-style teaching seminars and workshops. Finally, communication and feedback is considered essential in a technology as rapidly developing as that of microsequence array; thus regular user meetings are scheduled at least annually, and a close and frequent communication between the Core staff and the corporate company providers (Affymetrix, Agilent, Silicon Genetics, Partek) is maintained.
Significance of the Technology
A novel technology was developed recently for the determination of global mRNA expression profiles based on hybridization of entire native mRNA populations to high-density arrays of synthetic oligonucleotide gene probes. Broad gene expression profiles can be obtained for cells or tissue material within one time-saving and standardized experimental approach.
Microarray-based comparative analysis of global gene expression patterns on biological or pathologic material with different phenotypes will provide crucial information with regard to candidate genes or gene products which are involved in a disease process. Combined with sophisticated data analysis software tools, novel factors and/or regulatory events which contribute to phenotypic differences or changes in the cells can be identified which would otherwise not be accessible. Diagnostically relevant indicator gene sets can be discovered which are responsible for malignancy and phenotypic properties of tissues. The combination of microarray expression analysis with laser capture microdissection (LCM) is currently the most powerful approach to analyze gene expression in homogeneous cell populations derived from heterogeneous tissue material. Thus, this combination can be used to classify tissue subtypes according to their phenotypic and/or clinicopathologic differences. Moreover, microarray gene expression profiling may soon emerge as alternative for large-scale animal studies on toxicities, efficacies, and side effects of therapeutic agents to be tested.
Advantages and limitations: Experiments are time-saving, allowing global and parallel monitoring of gene expression. They provide quantitative comparisons which, after appropriate scaling, can be compared to outside data sets. Comparison with other public databases is possible (data-mining), with rapid expansions of such public databases, and rapid developments in engineering and software. On the other hand, the volume of accumulated data is clearly a challenge, and requires sophisticated software and statistical interpretations. With the most common nucleic acid hybridization-based arrays, no information on protein levels, activities, or modifications can be obtained, allowing a view only on the transcript expression and abundance levels. A certain level of technical wet-lab skills, careful design of experiments and controls are essential for a successful and meaningful analysis.
The UofL James Graham Brown Cancer Microarray Shared Facility has been operating since Spring 2001. Its service is centered around the currently most advanced and comprehensive array/instrumentation platform of high-density oligonucleotide array technology by Affymetrix, Inc. This platform allows RNA expression profiling, DNA mutation and single nucleotide polymorphism analyses, and re-sequencing procedures. The Facility is registered as Academic Core Account with Affymetrix, and is entitled to academic discount brackets for arrays and software licenses. Since its inception in Summer 2001, the Core has experienced a rapid growth in Core user numbers and diversity, allocated space, attendance in training events, and resources for data analysis and mining. There is considerable institutional interest in the application of the microarray technology to cancer molecular biology, tumor diagnostics and classification, and molecular target development. Most importantly, the Microarray Core has been an instrumental component in several funded and currently pending extramural grant applications, ensuring a potentially high pay-off for this investment. The Core is also an integral part of several multi-investigator Center and Training grants, like the active COBRE grants “Center of Biomedical Research Excellence in Molecular Targets” (D. Miller, P.I.) and “Molecular Determinants of Developmental Defects” (R. Greene, P.I.), the active T32 training grant “UofL Environmental Health Sciences Training Program” (D. Hein, P.I.), the pending P30 NIEHS “Center for Environmental Systems Biology” proposal (K. Ramos, P.I.) and the pending P42 “Early life exposure to hazardous waste substances” Superfund proposal (T. Knudsen, P.I.).
Qualifications of the Staff
Director: Dr. Wolfgang Zacharias, Ph.D. (Associate Professor, Dept. of Medicine and Dept of Pharmacology & Toxicology, Associate Scientist, James Graham Brown Cancer Center, UofL) is Director of the Facility. He is P.I. of several funded research projects (NIH/NIDCR RO1, Philip Morris External Research grant, Kentucky Lung Cancer Research grant), and has over four years of hands-on experience in all steps of microarray-based gene expression analyses with the Affymetrix platform. He was also participant in the NIH/NIDCR Consortium (PO1 DE12467) Harvard Forsyth Collaborative Microarray Core for oral cancer, and is co-author on several microarray-based manuscripts published or under review (see below). W.Z. has been an active molecular biology researcher for over two decades and has worked in the fields of DNA structure, gene expression analyses, and cancer molecular biology, with nearly 50 publications in peer-reviewed journal The Director is responsible for the financial oversight, personnel supervision and training, customer interactions, developmental and service decisions, liaison in business issues with Affymetrix, Inc, and Campus-wide training presentations.
Associate Director: Dr.Thomas C. Mitchell, Ph.D. (Assistant Professor, UofL Dept. of Microbiology and Immunology) functions as Associate Director for the Facility based on his prior experience and current interests in microarray technology including the Affymetrix platform. The Associate Director will replace the Director in periods of absence and also consult in administrative, directional or developmental decisions.
Research Technologist: A research technologist (Sabine Waigel, B.S.) is employed full-time as Facility Manager. She is well-trained in the purifications and analyses of nucleic acids, in basic molecular biological techniques, and is experienced in all aspects of the platform-specific sample preparation procedures. She also has extensive expertise in working with Windows-based software programs, networking aspects, and data analysis and archiving software. Ms. Waigel also has several years of administrative business experience in customer relations, invoicing and scheduling procedures. She is responsible for performing the instrument procedures and data acquisitions, distribution of data to users, perform or assist in sample preparation steps, and is in charge of instrument maintenance, record-keeping, billing, scheduling, and ordering.
Instrument upgrade: In Spring 2005, the instrument was upgraded with newest high-density Affymetrix Scanner 3000 model. This upgrade will allow processing of the newest generation of high-density v2.0 arrays with 8-5 mm feature size. Most importantly, it will also allow processing of the X3P array format with special probe design to interrogate RNA samples extracted from formalin-fixed paraffin-embedded tissues. Thus, this scanner upgrade will open new avenues to collect gene expression signatures not only from fresh tissues, but also from archived pre-existing tissue specimens.
Bioinformatics support: The new UofL Department of Bioinformatics & Biostatistics was established in Fall 2004 under the direction of Dr. Rudolph Parish, Ph.D., Professor of Bioinformatics, and is located in close proximity to the Core lab. This Department is bringing together the necessary personnel, resources, and logistics to ensure adequate and expert experimental designs and microarray data analyses for Core users. Two newly hired faculty with expertise in microarray data complexities and statistics will be available from July 2005.
Data management: In the process of expanding the Core resources, the development of a ftp-server-based database with online-based sign-up procedure is currently in progress as a collaboration with Dr. Ted Kalbfleisch, Director of Bioinformatics Operations for the Center for Genetics and Molecular Medicine.
Publications: Several manuscripts have been published recently which include microarray data collected in the Core (refs Kakar, Bodduluri, Partha, McMasters, Zacharias), and several are being reviewed or in preparation..
Facilities and Equipment
Location and space: The BCC Microarray Core lab is located in the recently opened Delia Baxter Research Building in the center of the UofL Health Science Center Campus. It occupies a wetlab (Room 323) of approx. 200 square feet. and an adjacent office (Room 321A) with workstations for data analysis and record keeping, and as technician office space. The Core is located within the two-building complex of the Donald Baxter and Delia Baxter Research Buildings, and in immediate proximity to the UofL Research Tower, the School of Dentistry, the James Graham Brown Cancer Center, and the UofL Hospital building complex; each of them only one block away.
Instrumentation: The Facility houses a complete Affymetrix Gene Expression Analysis instrument system for global RNA expression profiling and single nucleotide polymorphism (SNP) analysis capabilities. The Core lab includes the following components:
- The Affymetrix hybridization incubator Model 640 serves for overnight hybridizations of RNA samples to the microarray.
- The Affymetrix GeneChip Fluidics Station 450 is an upgraded multi-module, software-controlled component for the washing and staining steps of the hybridized array.
- The Affymetrx GeneArray Scanner 3000 confocal laser scanner measures hybridization intensities by quantitating fluorescence intensities of the labeled RNA sample across the array grid. This recently upgraded scanner is capable of scanning the high density 2.0 GeneChips containing up to 47,000 transcripts, as well as the Human X3P array for samples prepared from FFPE tissues.
- A Dell OptiPlex GX270 computer workstation, loaded with the Affymetrix Microarray Analysis Suite GCOS software package and internet connection, serves for data collection and analysis from the measured array scanner data; it also controls the automated steps on the fluidics station and scanner.
- An Agilent Bioanalyzer 2100 is set up for monitoring quality and quantity of very low amounts of cellular RNAs with sensitivity in the picogram range.
Software: Two additional computer workstations (Dell Optiplex GX 280, 350 GB hard drive, 2 GB RAM and Dell Optiplex GX 260, 100 GB hard drive, 1 GB RAM) with Affymetrix GCOS software packages and network connections are present in the Facility office for data analyses. The loaded operating software allows analyzing data by using different algorithms via MAS v5, RMA, or PLIER. For more sophisticated analyses of multi-component data sets, statistical analyses and graphic displays of filtered datasets, the Facility has annual licenses for GeneSpring v7.2 (Silicon Genetics, Redwood City, CA), PartekPro v6.05 (Partek Inc., St. Charles, MO), ArrayAssist v3.0 and PathwayAssist v3.0 (Stratagene, La Jolla, CA) data analysis software, statistics freeware program R with a module for micro-array analysis, and free access to the Bioconductor web resource; all are available to all users. For SNP analysis, the Affymetrix GDAS v3.0 software package can be installed.
Several opportunities for training at different levels are available through the BCC Microarray Core Lab. Most importantly, new Core users and their lab personnel will be trained by the Facility staff in the wetlab procedures for sample preparation, sample quality assessment procedures, and in basic data analyses. Detailed protocols and instructions for these steps are provided to the labs and, if necessary, the steps will be performed in the Facility lab under direct supervision by the technician. Also, classroom-style training events are scheduled and organized by the Core for UofL Health Science Center users on a regular basis. These include training in microarray technology concepts, lab procedures, data analysis and data quality determinations, and training in various software applications).
In addition, the Core Director participates in annual student course lecture training on microrray technology for graduate programs in Pharmacology & Toxicology (Course PHTX 665: Research Methods in Pharmacology and Toxicology; 4 hours) and Cell Biology (Course MBIO 667: Graduate Cell Biology, Gene array technology; 2 hours), Also, the Core Director is enrolled in several funded NIH and other extramural projects and pending applications, either as co-investigator or consultant for the microarray components of these projects.
For “training of the trainers”, frequent and regular communication with the instrumentation (Affymetrix) or software (Silicon Genetics, Partek) provider field specialists has been established and maintained over years. Either the Core Director (W.Z.) or the Core Manager (S.W.) has participated in the annual Affymetrix Core User Meetings.
User sign-up and scheduling: A Core website online sign-up procedure is currently in development and will be implemented in Summer 2005. This system will allow users to sign up for planned experiments, projects, and Core usage, and will store user information for scheduling, progress tracking, and subsequent billing and invoicing procedures. The plan is to simultaneously import parts of the user-entered information (date of sign-up, principal investigator and affiliations, experiment name, source of materials, sample and replicate sizes, array types, and funding sources) into the instrument experimental information files for status tracking and file identifications. This information will follow the proposed MIAME (minimum information about microarray experiments) guidelines (33), and will also be the basis for continuous monitoring of user frequencies, departmental affiliations, billing status, and funding sources of research projects.
Work flow: The Core places emphasis on ensuring upfront sound experimental design as well as expert data analysis and statistical evaluations at the outcome. Thus, initial discussions between users and bioinformatics personnel are facilitated to allow for a smooth workflow for final data analyses. The intermediate steps of sample preparation, hybrifizations, and data acquisitions are performed by the Core personnel with participation of the user lab personnel
Access to Resource and Capacity: Priority will be given to Brown Cancer Center investigators on a first-come-first-serve basis. Other investigators may have access to the instrument provided that a) they will cover all costs for reagents, arrays, instrument supplies, and service time, and b) their scheduling does not impede with the scheduling of the above defined users. Under certain circumstances, the Director may decide about altering priority access in case of upcoming proposal or other submission deadlines. One cycle of array processing includes overnight hybridization, ~ 3 hours fluidics station protocols, ~ 20 min/array scanning and absolute analysis, each for Test3 and subsequently full-scale array. Two such cycles of 8 arrays each will be feasible per week, bringing the maximum capacity to 16 arrays/week or approximately 800 arrays/year.
Management and dissemination of data: The collected data (DAT, CEL, CHP, and RPT files generated by the Affymetrix MAS 5.0 or GCOS software) will be handed to the investigator and also archived in the Facility as back-up. The absolute gene expression data (CHP files) and reduced gene lists, filtered according to user-specified criteria, will also be saved as Excel files and handed to the investigator. As a member of the Affymetrix Academic Core Program, the Microarray Core can enable access for users to the GCOS software for further data manipulations and analysis on the user’s office or lab desktop. Data storage and management issues are handled with assistance of the bioinformatics personnel (Dr. Ted Kalbfleisch, Assistant Professor, Department of Bioinformatics, as liaison and software collaborator).
New user recruitment: The Core places special emphasis on recruiting new users and making the microarray technology available to newly developing projects. This is done through annual pilot project competitions which have been held in the past 2 years (see below). In addition, Microarray Core User meetings and several regularly scheduled training seminars and lectures are held annually which are open to any new and interested investigators. For new users, individual one-on-one bench training in sample preparation and data analysis procedures is offered by the Core personnel, and web seminars on technology, data analysis, and data-mining are announced to the Campus-wide list of current and interested users.
A crucial function of the Core in new user recruitment is to provide logistics and consultation support for new grant proposal to obtain funds for microarray based approaches. The Core Director is currently listed as consultant or co-investigator for microarray support in 3 active grants, two of them from the NCI (McMasters, Chesney) and 14 pending grant applications, 5 of them to the NCI (Gupta, Zhou, Klinge, Galandiuk, Zacharias).
User Forum: Meetings among past, current, and future microarray users are held once or twice annually for exchange of experiences, trouble-shooting, expressions of needs, and updates in the technology itself.
A procedure for collecting user fees by intra-university transfer (IUT) was implemented in Dec. 2003 according to the list of fees for different services published on the Core website: http://www.louisville.edu/medschool/medicine/microarray/.
Several different units of service are defined to accommodate the different qualifications and technological expertise within the user community (below). They are based on the average technician time needed to provide such services, and include contributions to the annual costs for maintenance and upgrade contracts for instrument and software. BCC members receive a 20% discount on the published fees, except array and reagent prices. The fee/price list includes the following items:
- Fixed fees for defined procedural steps/technician labor time;
- Fixed reagents prices based on supplier prices;
- Prices for arrays based on the institutional discount bracket;
- Per hour fees for data mining and analysis assistance;
- Free access to analysis software for trained users
Microarray Core Facility Price Structure
- Agilent BioAnalyzer – $40/chip
- RNA/DNA/protein analysis – ($20/chip, if users order their own reagents; $10/chip for instrument run only)
- Hybridization only – $170/sample (user provides labeled fragmented cRNA and assist in subsequent steps, Core provides hybridization/wash/stain reagents, instrumentation, incl. scanning, raw data files in text or Excel format and access to data mining software)
- cDNA synthesis – $140/sample (includes reagents and sample processing)
- cRNA synthesis – $160/sample (includes reagents and sample processing)
- Test-Chip – $50/sample (scan, stain and analysis)
- Data Analysis – $80 / 4 hours
- Purchase of reagents – $125/sample. for cDNA and cRNA synthesis (both steps combined)
Other available alternatives for microarray-based experimentations
a) commercially available small-scale “macro-arrays”: Such membrane – or glass slide-based arrays are available from different vendors, but usually include only a few hundred gene targets (~ 500 genes on a ClonTech macroarray for ~$400/array compared to ~47,000 transcripts on one Affymetrix HG-U133Plus2.0 array for 4525/array). Thus, these macroarays are not as useful for global screening and discovery as the genome-wide comprehensive Affymetrix microarrays used in the Core.
b) Affymetrix platform services by other institutions: According to other academic Core websites, the prices for the UofL BCC Microarray Core are competitive and in the same range for total sample costs as for other academic institutions. Some corporate vendors offer array hybridization and processing, but for higher prices and without any support for downstream data analysis and mining.
c) in-house printed arrays. Such technology, although available on the market, is highly cost- and labor-intensive, since it requires special robotics equipment, enclosed dust-free housing, costs and storage space and recording software for probe material, and dedicated personnel for operating, maintenance, and record keeping.
Thus, the services offered by BCC Microarray Core on Campus are a very cost-efficient, logistically convenient, and user-supportive alternative.