Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

High-density oligonucleotide (oligo) arrays are a powerful tool for transcript profiling. Arrays based on GeneChip technology are amongst the most widely used, although GeneChip arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptorne of Brassica oleracea L., a species for which no GeneChip (R) array is available, using a GeneChip (R) array designed for Arabidopsis thaliana (L.) Heynh. Genornic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip (R) array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genornic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptorne analysis using a.cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P) stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68% of the available PM probes from the analysis but retained >96% of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip (R) array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affyrnetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptornic analyses of a wide range of plant and animal species in the absence of custom arrays.