Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2004
Publication Date: 9/7/2004
Citation: Alba, R., Fei, Z., Payton, P.R., Liu, Y., Debbie, P., Rose, J., Martin, G., Tanksley, S., Jahn, M., Giovannoni, J.J. 2004. Ests, cdna microarrays, and gene expression profiling: tools for dissecting plant physiology and development. Plant Journal. 36:697-714. Interpretive Summary: Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically ripening) and for comparative genomics by comparing tomato versus pepper fruit transcriptome activity.
Technical Abstract: A variety of experimental designs are possible for microarray analysis, most of which have been discussed in detail. For profiling gene expression during time-course studies or analyses of developmental transitions we have found the direct-sequential linear design and the direct-sequential loop design to be particularly useful. More recently, experimental designs for microarray analyses have begun to incorporate interspecies comparisons using arrays that originate from one of the genomes being investigated. Towards this objective, comparison of closely related species is most informative because artifacts stemming from sequence divergence are minimized. As an example of this type of interspecies comparison we have co-hybridized labeled cDNA populations derived from pepper pericarp (breaker stage fruit) and tomato pericarp (breaker stage fruit) to our TOM1 microarray (whose construction and features are also described here). Pepper loci showing increased transcript abundance in this experiment (compared to expression in equivalent tomato tissue) are of particular interest because this result can not be explained by differential hybridization due to sequence divergence. Verification was accomplished on a genomics scale via comparison with the tomato EST expression database (http://ted.bti.cornell.edu/).