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Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer

Abstract

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.

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Figure 1: Effects of oligonucleotide length and hybridization stringency on hybridization to tiled DNA oligonucleotide microarrays.
Figure 2: Dependence of hybridization intensity on number and location of mismatches and deletions.
Figure 3: Reliability of transcript ratios from ink-jet oligonucleotide microarrays and comparison of three sample amplification and labeling methods.
Figure 4: Agreement in transcript ratio measurements among a single oligonucleotide per gene, the average of eight oligonucleotides per gene, and cDNA microarrays.
Figure 5: Detection of gene regulation in human NB4 cells using a single oligonucleotide per gene.

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Acknowledgements

We thank C. Armour, A. Lee, and M. Tran for expert technical assistance; S. Collins for the NB4 cell line; J. Ledbetter for assistance with flow cytometry; and C. Roberts for critical evaluation of this manuscript.

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Correspondence to Peter S. Linsley.

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Hughes, T., Mao, M., Jones, A. et al. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol 19, 342–347 (2001). https://doi.org/10.1038/86730

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