title: Automated high-throughput probe production for DNA microarray analysis.
abstract: DNA microarrays have become an established tool for gene expression profiling. Construction of these microarrays using immobilized cDNAs is a common experimental strategy. However, this is extremely laborious, requiring the preparation of hundreds or thousands of cDNA probes. To minimize this initial bottleneck, we developed a comprehensive high-throughput robotic system to prepare DNA probes suitable for microarray analysis with minimal user intervention. We describe an automated system using the MultiPROBE Nucleic Acid Purification Workstation to provide the liquid handling and other functions needed to optimize this process. We were able to carry out fully automated plasmid cDNA isolation, PCR assay setup, and PCR purification and also to direct the characterization and tracking of DNA probes during processing. Protocols began with the initial preparation of a plasmid DNA archive of bacterial stocks in parallel 96-well plates (192 samples/run) and continued through to the dilution and reformatting of chip-ready DNA probes in 384-well format. These and other probe production procedures and additional instrument systems were used to process fully a set of mouse cDNA clones that were then validated by differential gene expression analysis.
authors.affiliation: Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, MS 6123, Oak Ridge, TN 37831, USA. hoytpr@ornl.gov
authors.forename: P R
authors.initials: PR
authors.lastname: Hoyt
authors.forename: L
authors.initials: L
authors.lastname: Tack
authors.forename: B H
authors.initials: BH
authors.lastname: Jones
authors.forename: J
authors.initials: J
authors.lastname: Van Dinther
authors.forename: S
authors.initials: S
authors.lastname: Staat
authors.forename: M J
authors.initials: MJ
authors.lastname: Doktycz
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D015342:DNA Probes / Q000096:biosynthesis / Q000138:chemical synthesis*; D018076:DNA, Complementary / Q000032:analysis*; D004867:Equipment Design; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D011786:Quality Control; D012371:Robotics / Q000295:instrumentation / Q000379:methods*
chemical_list: D015342:DNA Probes; D018076:DNA, Complementary