Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.
We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.
Rosetta Inpharmatics, Inc., 12040 115th Avenue NE, Kirkland, WA 98034, USA.
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Hughes
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Mao
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Jones
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Burchard
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Marton
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Shannon
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Lefkowitz
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Ziman
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Schelter
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Meyer
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Kobayashi
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Davis
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Dai
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Stephaniants
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Cavet
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Walker
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West
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Coffey
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Shoemaker
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Stoughton
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Blanchard
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Friend
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Linsley
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization / Q000379:methods*; D019169:Jurkat Cells; D020014:K562 Cells; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D018075:RNA, Complementary / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae; D012680:Sensitivity and Specificity; D013997:Time Factors; D014158:Transcription, Genetic; D014212:Tretinoin / Q000737:chemistry; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D009841:Oligonucleotides; D018075:RNA, Complementary; D012333:RNA, Messenger; D014212:Tretinoin