BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 Results Microarray design Due to limitations of ORF assignment for specific spots of the previously described whole genome microarray plat- form [14], we designed a more targeted and fully vali- dated array to address developmental regulation of the trafficking system in T. brucei. Major classes of proteins involved in membrane transport include small GTPases, vesicle coats proteins, kinases and phosphatases, motility factors mediating movement of vesicles along cytoskeletal elements, tethering factors for binding of vesicles to target membranes, SNARE proteins, ESCRTs and numerous other molecules involved in protein sorting and mem- brane deformation. The trypanosome genome was parsed extensively for such factors [38-41] and, based on these and additional searches, a subgenome microarray was constructed. The array also included putative ER chaper- ones, peptidases bearing an ER signal peptide and poten- tially involved in degradation within the trafficking system, homologues of kinases implicated in membrane traffic in other systems [18], sphingolipid biosynthesis enzymes with roles in exocytosis of glycosylphosphati- dylinositol (GPI)-anchored proteins, as well as trypano- some-specific surface antigens, e.g. BARP, CRAM, and the invariant surface glycoprotein family, or ISGs [42-44]. In addition we also included oligonucleotides against ESAG6 and ESAG7, which encode the trypanosome trans- ferrin receptor, a molecule reported to exhibit expression that is modulated by growth conditions [45]. 673 ORFs in the T. brucei genome with a potential role in membrane trafficking were identified, representing ~8% of the predicted protein coding potential. Representatives of most components of the major gene families are found in the T. brucei genome, but notable exceptions are the absence of several coat systems [46], a relatively small number of GTPases and associated factors [30], and the absence of BAR and SH2 domains; BAR domains are used extensively for membrane deformation in higher eukary- otes [47], while the absence of SH2 domains may indicate limited or unconventional signalling through protein tyrosine kinase pathways [40,48]. Apart from clustering of multicopy genes, there is no location bias for a specific chromosome or region within a chromosome for these ORFs [see Additional file 1]. An additional 123 ORFs were also included as a reference. The first array (v1.0), used for comparison of life stages and analysis of Rab5 isoform over-expression, comprised 694 oligonucleotides, 56 of which targeted reference ORFs. A modified array (v1.1), used for all other analyses, included an additional 49 oli- gonucleotides. The vast majority of oligonucelotides are gene-specific but some are expected to hybridise to multi- ple transcripts from multicopy genes [see Additional file 2]. Overview of differential expression between trypanosome life-stages We extracted RNA from exponentially growing cultures of T. brucei bloodstream and procyclic forms, labelled and competitively hybridised the cDNA to the microarray. As expected, the majority of genes were equivalently expressed in the two life stages, but a small cohort did present significant differential expression (Figure 1). The heat-map (Figure 1B) indicates that the data for the most significantly developmentally regulated genes are highly reproducible across all replicates (8 arrays, 4 replicate spots per array). To identify which mRNAs are differentially expressed, limma and fspma packages were used to interrogate the data [49-51]. For comparison with previous work [14], ratios above 2.5 (+/- 1.325, log2 scale) were considered highly significant. Using this threshold, 8% of the genes represented on the array are upregulated in BSF and 5% in PCF, but excluding the reference set these numbers reduce to 4% in BSF and 2% in PCF. Using a whole genome microarray, Brems and cowrokers found only 2% of trypanosome genes were developmentally regulated, and while different pre-processing steps, in both mRNA prep- aration and data analysis, make direct comparison diffi- cult, the present data suggest that the trafficking cohort likely exhibits bias in developmental regulation. There is also greater upregulation in BSFs compared to PCFs, con- sistent with increased activity in the trafficking system in bloodstream stages [10]. Brems et al. found 80 genes upregulated in PCF and 34 in BSF while, using the same threshold, the present analysis identifies 16 upregulated in PCF and 34 in BSF, from a much smaller cohort. These data suggest that significant transcriptional level remodel- ling of the membrane trafficking system accompanies life- cycle progression in trypanosomes. Strongly developmentally regulated genes include several reference ORFs with known regulated expression, i.e mRNAs for procyclin, trans-sialidase, CRAM, CAP17, and CAP5.5 are increased in PCF, while ESAG6, ESAG7, ISG65, ISG75, GPI-PLC, and CAP15 are upregulated in BSF (Table 1). These data are fully consistent with previ- ous work and support the validity of the microarray approach for identifying previously uncharacterised developmentally expressed genes. Rab11, also develop- mentally regulated [15], gave an expression ratio below 2.5 by both limma and fspma analysis. Hence, analysis using a more relaxed threshold to identify developmen- tally regulated mRNAs was also used. Table 1 lists all ORFs which either gave a ratio above 2.0 (+/- 1.00, log2 scale) by limma or fspma analysis, or which had a B-statistic value greater than 1.0 in limma, indicating statistical signifi- cance in differential expression, despite a low relative expression ratio. Excluding the reference set, 101 genes Page 3 of 28 (page number not for citation purposes)