Expression profiling using a hexamer-based universal microarray

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Abstract

We describe a transcriptional analysis platform consisting of a universal micro-array system (UMAS) combined with an enzymatic manipulation step that is capable of generating expression profiles from any organism without requiring a priori species-specific knowledge of transcript sequences. The transcriptome is converted to cDNA and processed with restriction endonucleases to generate low-complexity pools (80–120) of equal length DNA fragments. The resulting material is amplified and detected with the UMAS system, comprising all possible 4,096 (46) DNA hexamers. Ligation to the arrays yields thousands of 14-mer sequence tags. The compendium of signals from all pools in the array-of-universal arrays comprises a full-transcriptome expression profile. The technology was validated by analysis of the galactose response of Saccharomyces cerevisiae, and the resulting profiles showed excellent agreement with the literature and real-time PCR assays. The technology was also used to demonstrate expression profiling from a hybrid organism in a proof-of-concept experiment where a T-cell receptor gene was expressed in yeast.

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Figure 1: Schematic of the GenCompass process and the resulting targets.
Figure 2: Gene coverage and multiple tags per gene with UMAS technology.
Figure 3: Bp composition of ligation signatures and reproducibility across concentrations.
Figure 4: Determination of detection limit and system reproducibility.
Figure 5: Expression profiling of yeast in response to galactose induction.
Figure 6: Detection of a mouse transgene in a yeast background.

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Acknowledgements

The authors are grateful to Gavin Sherlock for expert technical advice; Nam-Hai Chua, Jon Morrow and Paul Kaplan for their scientific input and encouragement; Jose Lage, Grant Carlson and Gisela Carlson for comments on the manuscript; Christina Mityas, Mike Murtha, Yih-Woei Fridell and Shane Weber for their efforts in the early stages of this work; Tom Owen for technical assistance; and Meri Ross for manuscript preparation. We also thank all of our colleagues at Agilix for helpful discussions, continuing scientific input and excellent support.

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Correspondence to Craig E Parman or Paul M Lizardi.

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Competing interests

The Agilix employees who are listed as coauthors, and Junhyong Kim and Paul Lizardi (who are scientific advisors to the company), either have or are eligible for stock options through the company's stock option program.

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Roth, M., Feng, L., McConnell, K. et al. Expression profiling using a hexamer-based universal microarray. Nat Biotechnol 22, 418–426 (2004) doi:10.1038/nbt948

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