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 and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.
Highlights
High-density oligonucleotide arrays are a powerful and widely used tool for large-scale gene-expression profiling [1]
Perfect-match A. thaliana probes which hybridised to the B. oleracea genomic DNA above selected hybridisation intensities were selected for subsequent B. oleracea transcriptome analysis using probe mask files generated using a .cel file parser script
Probe-selection was tested by quantifying the transcriptional response of B. oleracea to a mineral nutrient stress using probe mask files generated at gDNA hybridisation intensity thresholds ranging from 0 to 1000
Summary
High-density oligonucleotide (oligo) arrays are a powerful and widely used tool for large-scale gene-expression profiling [1]. One extensively validated oligo array type is available as commercial GeneChip® technology (Affymetrix, Santa Clara, USA). GeneChip® arrays use probe-sets rather than single oligos per gene, comprising of between 11 and 20 probe-pairs to quantify abundance for each transcript. Transcript abundance can be calculated either from extrapolated hybridisation differences between PM and MM probes across a probe-set, or by using the PM data depending on the analysis used. An example of the wide-scale adoption of GeneChip® technology can be seen within the plant sciences research community, where data from thousands of GeneChip® arrays under large numbers of experimental challenges on the model plant Arabidopsis thaliana (L.) Heynh. At present, GeneChip® arrays are available for only a few species of eukaryotes. In contrast to A. thaliana, the transcriptomes of most agriculturally- or ecologicallyimportant plant species are less extensively studied, since extensive sequence information and the fabrication of expensive custom arrays would be required before experimentation can begin
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