biogem

About the Biomedical Genomics Core Facility (BIOGEM)

BIOGEM CORE Director: Gary Hardiman, Ph.D.

1. Objectives and Rationale for a Genomics CORE

The confluence of biotechnology, computer sciences and the completion of genome sequencing efforts for several organisms have resulted in revolutionary changes in how biomedical research can be done. It is now possible to synthesize high-density arrays of specified DNA sequences that, for example, include every known gene of an organism on a single glass slide or "chip". Labeled RNA or DNA targets (such as messenger RNAs obtained from cells, tissues or organisms under different conditions) can be analyzed by hybridization on the chip (1-5). This technology allows several types of questions to be asked at a qualitatively different scale than has been possible previously. A major goal in efforts to understand the mechanisms by which signal transduction pathways regulate programs of gene expression is to identify their direct target genes in response to regulatory signals. The BIOGEM CORE Facility provides expression services to help advance these efforts.

2. Description

History of the CORE

The Biomedical Genomics Microarray (BIOGEM) CORE Facility was established at UCSD in February 2000 to meet the needs of multiple laboratories interested in microarray technology.

Since becoming operational, four high-density cDNA microarrays have been fabricated and made available to the UCSD community; a mouse ~10K element array, a complete yeast genome array, a Drosophila ~6K element EST based array and a human ~10K element array. Additionally custom microarrays have been successfully printed from Dictyostelium discoideum and Arabidopsis clone sets. A custom murine cDNA microarray has been printed using clone sets for studies of metabolism, atherosclerosis and inflammation. Custom arrays have also been fabricated for to facilitate studies of the expression of Human Herpes Virus 8 (HHV8) genes in Kaposi's sarcoma patients. Numerous expression profiles have been generated to date using the high-density arrays described above and a record of accomplishment of generating high quality results has been established. Labeling methods have been investigated and optimized to perform microarray studies. Various slide chemistries have been thoroughly investigated and the best surfaces are currently used for all experimentation.

In September 2002 BIOGEM became the official UCSD site for processing of the commercial CodeLink bioarrays (from Amersham Biosciences). Since then we have added the Agilent and ABI platforms. The home-made cDNA microarrays have been retired in favor of these higher density and higher quality oligonucleotide arrays. The investigator typically provides RNA and BIOGEM will label the target, perform the hybridization and provide the user with raw and normalized microarray data. The CodeLink, Agilent and ABI systems are particularly useful when carrying out experiments with very small amounts of RNA. Utilization of Total RNA is beneficial for several reasons. First, it results in decreased time, labor and costs associated with not having to purify poly(A)+RNA. Second, many samples (biopsy, for example) do not contain sufficient amounts of total RNA to enable purification of poly(A)+RNA. Such samples could only be processed if the small amount of present total RNA could be used. Third, the less the biological sample is manipulated, the lower the chance that artifacts due to RNA processing will be introduced. Finally, purification of poly(A)+RNA, typically performed on an oligo(dT) column, can bias the sample by enriching for polyadenylated transcripts.

3. Facilities

Space

The BIOGEM facility is an 1800 square foot center in the Leichtag Biomedical Building. The two laboratories contain the equipment for the production and capture of microarray data. The robotics and DNA probe production lab is located in room 173, and this area houses the MicroGrid Arrayer, Axon and Agilent scanners and ASP in a semi-clean-room environment. This laboratory also houses the liquid handling robotics and the PCR machines. The molecular lab located in room 172 houses all RNA and cDNA preparation equipment as well as -20° C refrigerators. The microarray center includes two additional small rooms devoted to gel running and analysis and custom robotics development. Two -80° C freezers are located on the first floor of the Leichtag Biomedical Building. Room 172, houses four central computer servers, in addition to several data analysis workstations. The facility is centrally located on campus, in close proximity to the School of Medicine.

Equipment

BIOGEM is equipped with two functional microarray spotting instruments and two scanners. The MicroGrid2 (Genomic Solutions) uses up to 48 capillary pens to deposit nanoliter volumes of samples at high density onto metal coated and aminosilane coated glass slides. This spotter is the "work horse" apparatus in the facility, and is housed in the clean room environment in room 173, in order to keep the samples as clean as possible, and to minimize dust contamination of printed arrays. This spotter was acquired in 2003. The MicroGrid facilitates high density spotting of up to 44,000 spots per glass slide array with current slide chemistries. The gridding head can facilitate a maximum of 48 capillary pens, which deposit sub-nanoliter volumes. The MicroGrid spotting apparatus has an inbuilt humidity controller and refrigeration unit, which maintains the relative humidity at a constant level of 55%, minimizes evaporation, and enables optimal printing of microarrays. The built-in humidity control ensures optimum spot morphology. The spotter can print a 44K array in 36 hours.

In addition the facility has an Affymetrix arrayer (GMS417) and a home-made "Pat Brown" style spotter. The Affymetrix (formerly Genetic Microsystems) instrument is based on the pin-and ring technique. This technique involves dipping a small ring into the sample well and removing it to capture liquid in the ring. A solid pin is then pushed through the sample in the ring and sample trapped on the flat end of the pin is deposited onto the surface. Both arrayers are being used to extend the throughput of the facility, and are being used for spotting smaller custom protein arrays (up to 1000 probes).

BIOGEM has an Agilent scanner with an automatic slide loader and a proprietary lens that facilitates scanning of forty-eight arrays with high spatial resolution. In addition BIOGEM has an Axon 4000 scanner. Both scanners allows detection of sub-attomole amounts of fluorescent dyes (although dual color fluorescence using Cy3 and Cy5 fluors is the standard). These scanners allow BIOGEM to scan a wide variety of arrays, commercial and "in-house" that printed in different formats. The facility also has an Amersham Lucidea Automated Slide Processor (ASP) for hands-free hybridization and washing of up to 12 slides in a single run. For the application of microarray gene expression experiments, the instrument provides automated pre-treatment, non-static hybridization, and stringency washing of microarray slides. The hybridization solution is mixed during incubation to increase hybridization efficiency and improve reproducibility.

Microarray fabrication requires the availability of sufficient DNA probes, synthesized oligonucleotides or PCR amplicons prior to printing the array. A popular approach in recent years has been the generation of arrays from EST sets constructed in plasmid vectors. This involves replication of bacterial clones, amplification of many cDNA inserts, confirmation of the success of each PCR reaction, purification and dessication of the DNA and finally resuspension in 50% DMSO before printing. As these approaches are laborious and need to be performed routinely in a university CORE facility we sought to automate as many of these steps as possible.

For microarray probe preparation (i.e. the DNA printed on glass), BIOGEM has two PE Biosystems GeneAmp PCR machines and two MJ Research DNA Engine Tetrads. This machinery enables ten concurrent 96 well plate amplifications, and up to 1920 PCR amplifications to be performed per day.

BIOGEM has an Alpha Imager gel imaging system for digitized tracking of the array probes electrophoresed on agarose gels, ensuring that high quality probes are generated. Both fluorescently labeled microarray targets and DNA probes are quantified with a universal microplate spectrophotometer, and an FLX800 fluorimeter. This enables absorbance and fluorescence readings in a high throughput fashion. Additionally small RNA amounts are evaluated and quantified using an Agilent Bioanalyzer 2100. BIOGEM has a "speed-vac/refrigerated vapor trap system", designed for use with 96 and 384 microtiter well plates, in addition to microtubes. This allows rapid dessication of DNA following purification and avoids inconsistencies generated by ethanol precipitation.

Informatics Resources

Microarray informatics falls loosely into three distinct categories, clone and library management, spot finding or image acquisition, and finally data analysis and mining. The final component the data mining is performed independently by researchers or with assistance from BIOGEM personnel, depending on the level of experience of the investigator.

Using a MySQL database constructed in house, all images, sample information, reagent tracing and investigator information is centrally stored and archived.

Computer hardware resources include three central servers and twelve workstations. A Dell Poweredge server (2x833 mhz, 512 MB RAM, 2X80 GB SCSI hard drives) serves as a mass storage device, providing archiving and backup capabilities for raw data sets before they are imported into the central BIOGEM database. Additionally the Dell Poweredge server hosts the license files for the Biodiscovery software suite, enabling this software to be run in remote locations, thus allowing the researcher to access his or her data from their lab or laptop computer. A Sun Ultra 10 serves as a central internal facility database and web server. BIOGEM has constructed from two Intel based machines a load-balancing, clustered linux web server. Each machine is comprised of a Pentium IV (1.8 GHz) processor, 512 MB memory, redundant high-speed storage drive arrays, independent high-speed operating system drives, automatic battery back-up power and large swap space for very large file analysis.

Literature Cited

Brown, P.O., and D. Botstein. 1999. Exploring the new world of the genome with DNA microarrays. Nature Genet. 21:33-37.
Cheung, V.G., M. Morley, F. Aguilar, A. Massimi, R. Kucherlapati, and G. Childs. 1999. Making and reading microarrays. Nature Genet. 21:15-19.
Debouck, C., and P.N. Goodfellow. 1999. DNA microarrays in drug discovery and development. Nature Genet. 21:48-50.
Duggan, D.J., M. Bittner, Y. Chen, P. Meltzer, and J.M. Trent. 1999. Expression profiling using cDNA microarrays. Nature Genet. 21:10-14.
G. Hardiman. (2002). .Microarray Technologies.. Pharmacogenomics 3(3): 293-297.

Last Updated March 2008
By Dr. Gary Hardiman