BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 end of each target protein coding region. 694 oligonucle- otides were designed, targeting 796 genes (several ORFs are multicopy and hence multiple transcripts are sampled by a single oligonucleotide in such cases). The array encompassed 56 oligonucleotides targeting 123 reference ORFs, whose expression pattern has been established pre- viously. Oligonucleotides were manufactured by Illumina (Invitrogen) and included a 5' amino modification for attachment to Codelink activated slides (GE Healthcare). Arrays were printed using a BioRobotics Microgrid (Genomic Solutions) equipped with 16 NanoPins (Matrix Technology Corporation) in the Centre for Microarray Resources, Department of Pathology, University of Cam- bridge. Slides were prepared, washed and dried according to the manufacturer's instructions (GE Healthcare). Each oligonucleotide was spotted in quadruplicate, while α- and β-tubulin were spotted at the corners of the grid and reference oligonucleotides and additional spotting con- trols (empty, water, spotting buffer) were distributed ran- domly. Cells, in vitro and in vivo culture conditions Cultures of T. brucei brucei Lister 427 single marker blood- stream form (SMB) and procyclic form (PCF) strain 427 were maintained in HMI-9 (Invitrogen) and SDM-79 (Sigma) media, respectively [139-141]. Rab5A and Rab5B over-expressing PCF lines have been described previously [116]. A VSG knockdown line was generated by expres- sion of a double strand fragment of the 221 VSG ORF (PCR primers: TTTCTGCAGCGGTCACTATG and TGCTT- GTTGTTGGCTTTCAG) cloned into Eam1105I-digested p2T7TABlue [61]. Tetracycline-responsive SMB cells were transfected with NotI-digested p2T7TABlue-VSG by electro- poration, selected and maintained with 2.5 μg/ml G418 and 2.5 μg/ml hygromycin. RNAi was induced by adding 1 μg/ml tetracycline. The clathrin RNAi BSF line was gen- erated as described [114] except that the TbCLH gene frag- ment was subcloned into the p2T7TA vector [114] and an Amaxa nucleofector was used for transfection [142]. Transformants were maintained under 2.5 μg/ml neomy- cin and 2.5 μg/ml hygromycin selection, and RNAi was induced by adding 1 μg/ml tetracycline. For treatment with dithiothreitol (DTT) and tunicamycin, SMB cells were grown in HMI-9 to 1 × 106 cells/ml, and the media supplemented with either DTT (Sigma) at 1 mM, or tuni- camycin (Sigma) at 5 μg/ml. Samples for RNA extraction were taken after 1 hr and 4 hr for DTT addition and 4 hr and 24 hr for tunicamycin. To assess the influence of serum on gene expression, cells were grown in standard media until a concentration of 1 × 106 was reached and harvested by centrifugation at 800 g for 10 minutes. The cell pellet was re-suspended in media lacking fetal bovine serum (FBS), and supplemented with 5 mg/ml BSA with or without 0.3 mg/ml bovine holo-transferrin (Sigma), to simulate physiological concentrations of albumin and transferrin in media supplemented with 10% FBS [129]. For 30% FBS treatment cells were grown in HMI-9 media supplemented with 30% FBS. For in vivo grown cells, the blood from rats infected with monomorphic bloodstream forms (MITat1.1 at 1 × 109 cells/ml parasitemia) was taken by aortic puncture into heparinized syringe. The buffy coat fraction was prepared by centrifugation of infected blood (600 g, 10 min at 4°C). All subsequent treatments were performed at 4°C. Trypanosomes were purified from the buffy coat using PSG (NaH2PO4 3 mM, Na2HPO4 50 mM, NaCl 44 mM, sucrose 100 mM, adenosine 0.1 mM, pH 8.0). The cells were washed three times in PSG and then resuspended at 109 cells/ml. RNA extraction RNA was extracted using the Qiagen RNeasy kit following the manufacturer's instructions. 1 × 108 BSF (SMB) or 5 × 107 PCF cells were used per extraction. RNA quantity and quality was measured using a NanoDrop ND-1000 (Nan- oDrop Technologies) and denaturing formaldehyde agar- ose gels [143] prior to further analysis. For the MITat1.1 cells, RNA was prepared using the Absolutely RNA kit (Stratagene) according to the manufacturer's instructions. Array hybridization For each experiment, hybridisations were performed with at least three biological replicates with Cy3/Cy5 dye- swaps. Preparation of amplified and labelled cDNA tar- gets were essentially as described [144] except that Ampl- iTaq DNA polymerase Buffer II (Applied Biosystems) was used for the cDNA amplification. Following hybridisa- tion, slides were washed for 5 min in 2 × SSC, 5 min in 0.1 × SSC/0.1% SDS, 5 min in 0.1 × SSC and rinsed in dH2O. All washes were performed at room temperature. After washing, slides were dried by centrifugation and scanned on an Axon 4100 scanner using GenePix software (Molec- ular Devices). Fluorescence intensity was normalised so that the total signal for the Cy3 and the Cy5 channels were equivalent. Raw image data were extracted using Bluefuse (BlueGnome). Array data analysis To identify significant differential expression, the microar- ray data were analysed using limma (version 2.4.7) [50] from the Bioconductor open-source project [49] running under R (version 2.2.1), and a custom program written by K. Kelly (Cambridge). Data pre-processing comprised within-array print-tip Loess normalisation and between- array scale normalisation. To identify differentially expressed mRNAs a linear model was fitted to the data, using as weights the square root of the "confidence" value given by Bluefuse for each spot (which depends on the area, circularity and uniformity of the spot, signal to noise Page 22 of 28 (page number not for citation purposes)