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Generation of an oligonucleotide array for analysis of gene expression in Chlamydomonas reinhardtii

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Abstract

The availability of genome sequences makes it possible to develop microarrays that can be used for profiling gene expression over developmental time, as organisms respond to environmental challenges, and for comparison between wild-type and mutant strains under various conditions. The desired characteristics of microarrays (intense signals, hybridization specificity and extensive coverage of the transcriptome) were not fully met by the previous Chlamydomonas reinhardtii microarray: probes derived from cDNA sequences (~300 bp) were prone to some nonspecific cross-hybridization and coverage of the transcriptome was only ~20%. The near completion of the C. reinhardtii nuclear genome sequence and the availability of extensive cDNA information have made it feasible to improve upon these aspects. After developing a protocol for selecting a high-quality unigene set representing all known expressed sequences, oligonucleotides were designed and a microarray with ~10,000 unique array elements (~70 bp) covering 87% of the known transcriptome was developed. This microarray will enable researchers to generate a global view of gene expression in C. reinhardtii. Furthermore, the detailed description of the protocol for selecting a unigene set and the design of oligonucleotides may be of interest for laboratories interested in developing microarrays for organisms whose genome sequences are not yet completed (but are nearing completion).

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Acknowledgments

We would like to thank Michael Fero, Elena Seraia and John Coller of the SFGF Laboratory at Stanford for technical advice, printing the arrays and preparing the .gal file in a timely and expert manner. Furthermore, the development of the microarray presented in this manuscript could not have been accomplished without the major sequencing effort of the C. reinhardtii genome that was performed by the Joint Genome Institute (Walnut Creek, CA, USA). This work was supported by NSF Grant MCB 0235878 awarded to ARG. The authors would also like to thank Wirulda Pootakham and Olivier Vallon for helpful discussions and Erika Schraner for help with Supplemental Table 3.

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Authors and Affiliations

  1. Department of Plant Biology, The Carnegie Institution, 260 Panama Street, Stanford, CA, 94305, USA

    Stephan Eberhard, Monica Jain, Chung Soon Im, Steve Pollock, Jeff Shrager & Arthur R. Grossman

  2. Laboratoire de Physiologie Moléculaire et Membranaire du Chloroplaste, Institut de Biologie Physico-Chimique, UMR7141 (CNRS – Université Paris VI), 13 Rue Pierre et Marie Curie, 75005, Paris, France

    Stephan Eberhard

  3. Integrated DNA Technologies, 1710 Commercial Park, Coralville, IA, 52241, USA

    Yuan Lin & Andrew S. Peek

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  1. Stephan Eberhard
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  2. Monica Jain
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Correspondence to Stephan Eberhard.

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Communicated by D. Stern

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Eberhard, S., Jain, M., Im, C.S. et al. Generation of an oligonucleotide array for analysis of gene expression in Chlamydomonas reinhardtii . Curr Genet 49, 106–124 (2006). https://doi.org/10.1007/s00294-005-0041-2

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