Cotton spotted oligonucleotide microarrays for gene expression analysis

Microarrays offer a powerful tool for diverse applications plant biology and crop improvement. Recently, a global assembly of cotton ESTs was constructed based on three Gossypium. Using that assembly as a template, we now describe the design and creation and of a publicly available oligonucleotide array for cotton, useful for all four of the cultivated species. Synthetic oligonucleotide probes were generated from exemplar sequences of a global assembly of more than 150,000 cotton ESTs derived from 30 different cDNA libraries representing many different tissue types and tissue treatments. A total of 13,158 oligonucleotide probes are included on the arrays, optimized to target the diversity of the transcriptome but also including previously studied cotton genes, duplicated gene pairs derived from a paleoduplication event, transcription factors, and homology to protein coding genes in Arabidopsis. About 10% of the oligonucleotides target unidentified protein coding sequences, thereby providing an element of gene discovery. Because many oligonucleotides were based on ESTs from fiber-specific cDNA libraries, the array has direct application for analysis of the fiber transcriptome. To illustrate the utility of the array, we hybridized labeled bud and leaf cDNAs from G. hirsutum and demonstrate technical consistency of results. The cotton microarray provides a reproducible platform for transcription profiling in cotton, and is made publicly available through http://cottonevolution.info. Keywords: self vs. self; platform testing Three replications of leaf and bud RNA were collected from the 4-5 plants grown in green houses at Iowa State University. RNA was separately extracted and pooled into a single 'leaf' and 'bud' sample. These pooled RNA samples were then amplified (TargetAmp, Epicentre) and labelled with Cy3 and Cy5. The bud samples were split into two so as to minimize any error that might arise from something besides the microarray. Replication bud vs. bud' and leaf vs. bud experiments were carried out. Data were analyzed in R and the results are present to the research community documenting the availability of a cotton oligonucleotide microarray.