TRANSCRIPTOME 2002: From Functional Genomics to Systems
Development and Production of a Redundant Oligonucleotide Musclechip: Use for Verification and Prioritization of ESTs
Rehannah HA Borup1, Stefano Troppo2, Yi-Wen Chen1, Tanya M Teslovich1, Gerolamo Lanfranchi2, Giorgio Valle2, and Eric P Hoffman1, 1Research Center for Genetic Medicine, Children’s National Medical Center, Washington, DC, 2CRIBI Institute, University of Padova, Padova, ITALY
We describe the development, validation, and use of a highly redundant oligonucleotide microarray (MuscleChip) containing 4,601 muscle probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle sequencing project (28,074 EST sequences). This set included 452 novel EST clusters showing no match to previously characterized genes in any database. Each probe set was designed to contain 20-32 25mer oligonucleotides, with each probe evaluated for hybridization kinetics and similarity to other sequences. Approximately 120,000 oligonucleotides were synthesiz-ed. Hybridization of normal muscle cRNA to this MuscleChip showed a correlation between EST cluster member number (n-EST), and determination of a “present” call based on hybridization patterns to probe sets: 30% of singletons, 46% of duplex, and 66% of triplex EST clusters were identified as “present” in normal muscle. Limiting the analysis to the 452 novel EST clusters, and to _at probe sets (288 unique probe sets fulfilling design rules), we found 181/452 (40%) of the novel clusters to be expressed in normal muscle, thereby verifying these ESTs as expressed genes. A hyperlink table was developed to map each of these novel “present” calls confirmed hypothetical proteins to either the RefSeq database or genomic data.
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