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Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing

John Castle†, Phil Garrett-Engele†, Christopher D Armour, Sven J Duenwald, Patrick M Loerch, Michael R Meyer, Eric E Schadt, Roland Stoughton, Mark L Parrish, Daniel D Shoemaker and Jason M Johnson*

Author Affiliations

Rosetta Inpharmatics, Merck & Co. Inc., 12040 115th Ave NE, Kirkland, Washington 98034, USA

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Genome Biology 2003, 4:R66 doi:10.1186/gb-2003-4-10-r66

Published: 19 September 2003

Abstract

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.