By interrogating the transcriptome of E2Fa-DPa OE plants, which display ectopic cell division and endoreduplication, with ATH1 microarrays, we had identified 181 potentially direct E2F target genes, with many of them encoding proteins involved in cell cycle progression, DNA replication and chromatin dynamics ( Vandepoele et al., 2005 Vlieghe et al., 2003). To this end, we have previously generated plants ectopically overproducing the heterodimer E2Fa-DPa ( E2Fa-DPa OE De Veylder et al., 2002 Kosugi and Ohashi, 2003). A complete understanding of the role of the different E2F isoforms requires the comprehensive identification of their target genes. The A. thaliana genome encodes three E2Fs (E2Fa, E2Fb and E2Fc De Veylder et al., 2007), which are active in association with the dimerization partners DPa or DPb. E2Fs are conserved regulators of S phase-specific genes ( Blais and Dynlacht, 2007 Iaquinta and Lees, 2007). Thus, to analyze the expression of all genes, the Tiling 1.0R array needs to be hybridized with double-stranded cDNA.įor several years we have been interested in understanding the role of E2F transcription factors in regulating the plant cell cycle and plant growth. A further potential advantage of the ATH1 array is that probes are all antisense to the annotated transcripts, whereas probes on the Tiling 1.0R are all from one strand of the genome only, such that half of all genes will be represented by antisense probes, and the other half will be represented by sense probes. In addition to trying to avoid cross hybridization with other genes, probes were chosen such that their hybridization characteristics were as similar as possible. In contrast, the Arabidopsis ATH1 array contains sets of 11 25-mer probes that were designed to uniquely cover 3′ exons of A. thaliana genes, based on the now outdated TIGR 3.0 annotation ( Redman et al., 2004). Because of the design, only very limited consideration could be given to probe behavior in hybridization. The basis for the design was the NCBI Arabidopsis genome assembly (version 5), and mitochondrial and chloroplast sequences were included as well (NCBI accession numbers NC_001284 and NC_000932). The 25-mer probes are spaced (on average) 35 bases apart, as measured from the central position of adjacent 25-mer oligonucleotides, leaving a gap of 10 bases between adjacent probes ( ). The GeneChip ® Arabidopsis Tiling 1.0R array, commercially available from Affymetrix, is a single array with over 3.2 million perfect-match and mismatch (PM/MM) probe pairs that are tiled across the complete non-repetitive Arabidopsis thaliana genome. Whereas focused gene expression microarrays seek to measure the relative abundance of transcripts derived from a specifically targeted set of annotated sequences, tiling arrays can also be used, for example, to discover transcribed genomic regions that are independent of previous annotations, to detect non-coding RNA transcripts or to identify alternative RNA isoforms of known genes ( Bertone et al., 2004 Kapranov et al., 2002). These arrays are useful for several purposes, and can be used to analyze DNA content, as well as mRNA content. Probes on tiling arrays either partially overlap one another (true tiling) or are spaced at regular intervals. Recent advances in microarray technologies have resulted in the commercial availability of tiling arrays, making it feasible to interrogate whole genomes in an unbiased way. The latter groups increase the number of excellent candidates for new, direct E2F targets by almost twofold, from 181 to 334. With the tiling arrays we could identify 368 new E2F-regulated genes, with a large fraction including an E2F motif in the promoter. We show that with the appropriate data pre-processing, the estimated changes per gene for those with significantly different expression levels is very similar for the two array types. As a test case, we compared the transcriptome of wild-type plants with that of transgenic plants overproducing the heterodimeric E2Fa-DPa transcription factor. We also propose a method for selecting unique tiling probes for each annotated gene or transcript in the most current genome annotation, TAIR7, generating Chip Definition Files for the Tiling 1.0R array. Here, we present an approach to exploit this platform for quantitative mRNA expression analysis, and compare the results with those obtained using ATH1 arrays. Recently, Affymetrix has introduced its Arabidopsis Tiling 1.0R array, which offers whole-genome coverage of the sequenced Col-0 reference strain. The Affymetrix ATH1 array provides a robust standard tool for transcriptome analysis, but unfortunately does not represent all of the transcribed genes in Arabidopsis thaliana.