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Mentions: Arabidopsis thaliana PM probes hybridised extensively to the B. oleracea genomic DNA (Figure 1). When the gDNA hybridisation intensity threshold was increased from 0 to 1000 during probe mask file generation, PM probe retention in the probe mask files decreased rapidly. However, the retention of whole probe-sets, representing transcripts, were less sensitive to increases in gDNA hybridisation intensities during probe mask file generation, since only a minimum of one PM probe was required to retain a probe-set. For example, although the probe mask file generated using a gDNA hybridisation intensity threshold of 300 masked > 50 % of all PM probes, 98.8 % of available A. thaliana probe-sets were retained.
Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species
Bottom Line: Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip array.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 array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold.
Affiliation: Nottingham Arabidopsis Stock Centre, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK.
Abstract: 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 transcriptome of Brassica oleracea L., a species for which no GeneChip array is available, using a GeneChip array designed for Arabidopsis thaliana (L.) Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptome 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 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://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.
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