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

Hammond, J. P. ORCID: https://orcid.org/0000-0002-6241-3551, Broadley, M. R., Craigon, D. J., Higgins, J., Emmerson, Z. F., Townsend, H. J., White, P. J. and May, S. T. (2005) Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species. Plant Methods, 1. 10. ISSN 1746-4811 doi: 10.1186/1746-4811-1-10

Abstract/Summary

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/ webcite and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.

Item Type	Article
URI	https://reading-clone.eprints-hosting.org/id/eprint/33861
Identification Number/DOI	10.1186/1746-4811-1-10
Refereed	Yes
Divisions	Interdisciplinary centres and themes > Centre for Food Security Life Sciences > School of Agriculture, Policy and Development > Department of Crop Science
Uncontrolled Keywords	Arabidopsis thaliana Brassica oleracea Brassicaceae cross-species microarray oligonucleotide phosphate phosphorus plant nutrition probe masking transcriptomics
Publisher	BioMed Central
Download/View statistics	View download statistics for this item