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Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species
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Hammond, John P., Broadley, Martin R., Craigon, David J., Higgins, Janet, Emmerson, Zoe F., Townsend, Henrik J., White, Philip J. and May, Sean T. (2005) Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species. Plant Methods, Vol.1 (No.10). doi:10.1186/1746-4811-1-10 ISSN 1746-4811.
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Official URL: http://dx.doi.org/10.1186/1746-4811-1-10
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/ 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: | Journal Article | ||||
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Subjects: | S Agriculture > SB Plant culture Q Science > QR Microbiology |
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) > Warwick HRI (2004-2010) | ||||
Library of Congress Subject Headings (LCSH): | Oligonucleotides, Plant genome mapping, Gene mapping | ||||
Journal or Publication Title: | Plant Methods | ||||
Publisher: | BioMed Central Ltd. | ||||
ISSN: | 1746-4811 | ||||
Official Date: | 9 November 2005 | ||||
Dates: |
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Volume: | Vol.1 | ||||
Number: | No.10 | ||||
DOI: | 10.1186/1746-4811-1-10 | ||||
Status: | Peer Reviewed | ||||
Access rights to Published version: | Open Access (Creative Commons) | ||||
Funder: | Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC), Great Britain. Dept. for Environment, Food & Rural Affairs (DEFRA), University of Nottingham | ||||
Grant number: | HH3501SFV (DEFRA), HH3504SPO (DEFRA), IGF12422 (BBSRC) |
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