BMC Genomics 2006, 7:42 http://www.biomedcentral.com/1471-2164/7/42 with a 100 µm spot size. After printing the slides were UV irradiated to cross-link the DNA to the polylysine coating. Slide hybridisation, scanning and normalisation Slides were prehybridised by incubation for 45 min at 42°C in 50 ml of 5 X SSC, 0.1% SDS, 1% BSA. They were then rinsed twice in deionised water, once in isopropanol and dried. The labelled cDNA was denatured by heating at 95°C and then combined with 50 µl SlideHyb #1 (Ambion) and applied to slides. Slides were hybridised at 54°C for 16 hours in humidified chambers (Monterey). Post-hybridisation the slides were washed in the dark at 54°C for 10 min in 2 X SSC, 0.1% SDS, 5 min in 1 X SSC and 10 min in 0.1 X SSC. All buffers were filtered through 0.22 µm filters. The slides were dried and scanned in a ScanArray 5000 (Packard Biosciences) and the dual images collected in TIFF format. The combination and processing of the images was carried out using GenePix Pro (Axon Instruments). The data for each slide was nor- malised following the procedure of Baird et al., [71]. For each EST on the array, the normalised data from all 16 slides was combined and a number of average statistics calculated. ESTs were excluded from further analysis if they had more than 6/16 bad spots. The remaining ESTs were sorted based on the modified T value [72] for the log ratio of the mean [Additional file 3]. ESTs where the tail of the probability plot was more extreme than the 95% con- fidence limit [73] were counted as differentially expressed (Figure 1). All the microarray information has been sub- mitted into the National Centre for Biotechnology Infor- mation (NCBI) Gene Expression Omnibus (GEO) website [74]. The accession number for the experiment series is GSE3738. Northern blotting 20 µg of total RNA from duodenum tissue was separated on a 1% formaldehyde agarose gel and transferred to Hybond nylon membrane (Amersham) by capillary trans- fer. Probes were generated by amplification from the orig- inal EST using the gene specific primers listed in Table 5. The PCR products were verified by sequencing, labelled with α32P-dCTP (Amersham) using the RadPrime DNA labelling system (Invitrogen) and unincorporated radio- activity removed using the High Pure PCR product purifi- cation kit (Roche). Membranes were prehybridised for 45 min at 42°C in 10 ml ULTRAhyb (Ambion). The probes were diluted 1:10 in 10 mM EDTA and denatured by boil- ing for 10 min followed by cooling on ice for 5 min. After addition of 0.5 ml of ULTRAhyb, the probe was added to the prehyb solution and incubated overnight at 42°C. The membranes were then washed twice for 5 min at room temperature in 2 X SSC, 0.1% SDS followed by 2 X 15 min in 0.1 X SSC, 0.1% SDS at 42°C. The membranes were then exposed to BioMax XAR film (Kodak). The images were scanned using an ImageScanner (Amersham) and the resulting images quantitated using ImageQuant TL (Amersham) and normalised to GAPDH mRNA levels. This gene was not differentially expressed in the microar- ray experiment making it a suitable choice of housekeep- ing gene for normalisation. Microarray data interpretation All ESTs in the ovine libraries, along with all ovine ESTs deposited in NCBI were assembled into contigs using CAP3 [75] after an initial clustering step using BLAST. ESTs on the array were annotated by finding the human RefSeq (RefSeq release as at 11/4/2005) corresponding to the contig to which they belonged using BLASTN [76] and the following options: -e 0.01 -v 5 -b 5. Each EST was annotated with the top human RefSeq hit. In the case of the differentially expressed ESTs, a more stringent E value cutoff of 1 × 10-18 was applied. In cases where the EST matched more than one transcript variant of a gene then the top hit is listed. All analysis hereafter refers to the annotated RefSeq genes. Gene Ontology (GO) terms sig- nificantly associated with the differentially expressed genes were found using the Expression Analysis System- atic Explorer (EASE). The list of differentially expressed genes was submitted to EASEonline [77]. The background list submitted included all human RefSeqs on the array. EASE calculates overrepresented functional gene catego- ries compared to all the genes on the array [31]. A region of 1,500 bp upstream of the transcription start site of each of the RefSeq genes on the array was retrieved from the human genome browser at University of Califor- nia-Santa Cruz [78] and sequences were masked for repeats through the retrieval process. The choice of pro- moter length was based on the report that approximately 75% of human core promoters lie within 1,500 bp of the transcription start site [79]. Of the 9,238 unique RefSeqs on the array, promoter regions for 8,809 RefSeqs were retrieved. Motifs in the promoter regions of the differen- tially expressed RefSeq genes were detected using the MEME motif detection program [32]. The motif lengths ranged between 6 and 12 bases, any number of repetitions of the motif was permitted, the reverse complement was allowed and the top 15 motifs were identified in each group The identified motifs were also compared to the transcription factor database, TRANSFAC release 8.1, [33] using the method described in Aerts et al., [80]. Each motif was permuted 1,000 times and best matches and their level of significance are reported. The full list of motifs identified in the promoter regions of the genes more highly expressed in susceptible and resistant animals, and their top TRANSFAC hits are available in Additional files 1 and 2 respectively. In order to carry out a MAST analysis [81] motifs with a Pearson's correlation coefficient greater than 0.6 were identified and excluded from further analy- sis. Promoter regions of both groups of differentially Page 9 of 12 (page number not for citation purposes)