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Microarray Technology and Informatics
European Summer School - Microarray technology and Bioinformatics
Microarray Technologies - An Overview
Microarray Related Seminars
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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.
The Biomedical Genomics laboratory (BIOGEM) CORE Facility was established at UCSD in February 2000 to meet the needs of multiple laboratories interested in microarray technology.
The initial focus of BIOGEM was on fabricating high density cDNA and oligonucleotide microarrays. This technology has been retired in favor of superior commercial array platforms. Presently BIOGEM supports the Agilent and Illumina BeadArray platforms.
The investigator typically provides RNA and BIOGEM validates the starting total RNA, labels the target, perform the hybridization and provide the user with raw and normalized microarray data. The Illumina and Agilent systems are particularly useful when carrying out experiments with small amounts of total RNA (50 ng). The total RNA is amplified using an isothermal process to generate fluorescent cRNA targets for hybridization.
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 of introducing artifacts. Finally, purification of poly(A)+RNA, typically performed on an oligo(dT) column, can bias the sample by enriching for polyadenylated transcripts.
In January 2008, an Illumina 1G Analyzer was added to the Core. In February 2009, two additional Illumina 1G Analyzers were purchased by UCSD. These shared instruments have expanded further the sequencing capabilities of BIOGEM.
Because of its highly skilled personnel and range of instrumentation, BIOGEM has worked closely with individual investigators to fabricate custom arrays, as well as provide off-the-shelf microarrays. BIOGEM has also frequently been used as a beta testing site for new technology platforms, facilitating incorporation of these technologies as core services for the UCSD community.
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 in a semi-clean-room environment. This laboratory also houses 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. Two -80° C freezers are located on the first floor of the Leichtag Biomedical Building. The facility is centrally located on campus, in close proximity to the School of Medicine.
BIOGEM Facility equipment includes:
BIOGEM is equipped with a 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.
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.
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.
Microarray informatics falls loosely into three distinct categories, experiment design, wet lab experimentation, and finally data analysis and mining. BIOGEM assist with experimental design typically ensuring that there are adequate numbers of biological replicate samples. All the wet lab experimentation is carried out by BIOGEM personnel. 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 incoming sample and investigator information is centrally stored and archived.
Computer hardware resources include a centralized server called Microarrays and multiple workstations.
Last Updated March 2009
By Dr. Gary Hardiman
