CDNA clones and their application for cDNA microarray analysis

Currently there are nearly 23 million expressed sequence tags (ESTs) in the NCBI public collection as of August 2004, about 4 million of which derive from plants ( With many large-scale EST sequencing projects in progress and new projects being initiated, the number of ESTs in the public domain will continue to increase in the coming years. cDNA clones are a useful tool for expression profiling studies, because cDNA microarrays can be prepared directly from the isolated clones.

Altogether, 19 RIKEN Arabidopsis full-length (RAFL) cDNA libraries from plants grown under different conditions were constructed as reported previously (4, 35, 36). We have isolated 245 946 RAFL cDNA clones and they are clustered into about 18 000 non-redundant cDNA groups, covering about 70% of predicted genes (36; Seki et al., unpublished results).

The RAFL cDNAs were used for microarray analysis of expression profiles of Arabidopsis genes (5, 6) under various stress conditions, such as drought, cold and high salinity (9, 10), various treatment conditions, such as abscisic acid (ABA) (37), rehydration treatment after dehydration (18), ethylene (16), jasmonic acid (JA) (16), salicylic acid (SA) (16), reactive oxygen species-(ROS-) inducing compounds such as paraquat and rose bengal (16), UV-C (16), and inoculation with a pathogen (16, 17). We have also studied the expression profiles in various mutants and transgenic plants (9, 16, 38, 39). These studies have shown that cDNA microarray analysis is useful for analyzing the expression pattern of plant genes under various stress and hormone treatments, to identify target genes of transcription factors involved in stress or hormone signal transduction pathways, and to identify potential cis-acting DNA elements by combining the expression data with genomic sequence data. cDNA microarrays can be used in closely related species with about 90% sequence homologies of coding regions, such as rice and barley (40), and Arabidopsis and Thellungiella halophila (41).

In this chapter, we describe the protocol of our RAFL cDNA microarray analysis.

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