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Marc Goldstein, Beckman Coulter, Inc. The analysis of gene expression by hybridization to gene chips (Affymetrix) requires preparation of target RNA from total RNA isolated from biological samples of both control and test material. The quality of the output data is only as good as the material applied to the gene array, hence the need for a robust method without bias among the expressed genes. Automation is brought to bear on this process when the sample number exceeds what can be reliably accomplished using manual methods. Affymetrix has developed a High Throughput Array (HTA) system based on gene arrays in the bottom of each well of a 96-well plate. The Project Management team was brought in to enable the automation to process the large number of samples required to support the HTA system. The team developed a solution, based on the first custom solution at Affymetrix that provided increased throughput and capacity, as shown below. Starting with total RNA from a test or control biological sample (cell line or tissue) the automation takes the material through three methods, (1) cDNA synthesis (first and second strand synthesis) in the Tetrad2 thermal cycler, cDNA cleanup via filtration, (2) in vitro transcription for amplification and incorporation of a biotin label into the cRNA (target RNA), cRNA cleanup via filtration then (3) quantitation and normalization of the target RNA using a SPECTRAmax* (Molecular Devices) followed by hybridization on the HTA plate. The results of the hybridization are read on the Axon* scanner (Affymetrix). The team developed a highly custom automated system made up of the "best of breed" or most appropriate devices to meet the functionality, reliability and performance needs of the application. Cost was a secondary issue in this case. The custom components included the PE Flex Drop* for the dispense of a variety of reagents to the plates during the course of the process. The Tetrad2 thermal cycler, the core compliant ABGene ALPS-300 sealer and the custom ASP-50 piercer enabled online thermal cycling during the cDNA synthesis step. Pierced plates were held onto the deck in the custom plate flattening ALP (Automated Labware Positioner) as described in the November 2004 issue of T3 Update, to allow for precise, uniform pipetting operations with the Multichannel head. Heated and cooled Cytomat2 units allowed for proper temperature control during incubations. An on-deck custom Peltier ALP (described in the November 2004 issue of T3 Update) was used to keep reagents cool while on deck. The liquid handling portions of this application were particularly challenging and required a lot of input from the applications laboratory. The challenges have been met and now result in high-quality data output. Though designed for target RNA preparation from total RNA, this highly complex system could be used for just about any molecular biology task required. For simpler applications, this automated solution is over-engineered. * All trademarks are the property of their respective owners. For Research Use Only; not for use in diagnostic procedures. For comments or questions about T3 Update, please contact David Daniels, Ph.D., editor.
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