Accessibility navigation


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

Hammond, J. P., 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

[img]
Preview
Text - Published Version
· Available under License Creative Commons Attribution.
· Please see our End User Agreement before downloading.

763kB

It is advisable to refer to the publisher's version if you intend to cite from this work. See Guidance on citing.

To link to this item 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
Refereed:Yes
Divisions:Interdisciplinary centres and themes > Centre for Food Security
Faculty of Life Sciences > School of Agriculture, Policy and Development > Biodiversity, Crops and Agroecosystems Division > Crops Research Group
ID Code:33861
Uncontrolled Keywords:Arabidopsis thaliana Brassica oleracea Brassicaceae cross-species microarray oligonucleotide phosphate phosphorus plant nutrition probe masking transcriptomics
Publisher:BioMed Central

Downloads

Downloads per month over past year

University Staff: Request a correction | Centaur Editors: Update this record

Page navigation