BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 ratio, signal saturation, and the consistency of the Cy3/ Cy5 ratio in different pixels across the spot). Adjustment for multiple testing was carried out using the false discov- ery rate (FDR) method [145] in limma. For clustering analysis, data from the Bluefuse analysis were processed using the Gene Expression Pattern Analysis Suite (GEPAS) [146,147] using the DNMAD and SOTARRAY programs. Replicates were merged after filtering replicates with val- ues > 0.5 – 1.0 above the median of replicates. Hierarchi- cal clustering was performed using UPGMA and Euclidian distance with settings at normal. Quantitative real time (qRT)-PCR For cDNA synthesis, 2 μg RNA was diluted to 10 μl with diethylpyrocarbonate (DEPC)-treated water and dena- tured at 70°C, 5 min. 15 μl of a reaction mix was added (2.5 μl dNTPs (25 mM stock), 5 μl 5× reverse transcription buffer (Invitrogen), 2 μl 100 mM DTT, 0.5 μl RNAseOUT (recombinant ribonuclease inhibitor, 40 U/μl, Invitro- gen), 2 μl oligo dT (T30VN, 10 μM stock), 0.5 μl Super- script II Reverse Transcriptase (200 U/μl Invitrogen), and 2.5 μl DEPC-treated water) and incubated at 37°C for 1 hr, heat-inactivated at 90°C, 5 min and finally diluted to 200 μl with DEPC-treated water. For qRT-PCR, 5 μl of cDNA was used in a 25 μl reaction including IQ SYBR Green Supermix (BioRad) with 0.4 μM gene-specific forward and reverse primers [see Additional file 2]. qRT-PCR reactions were performed in white thin- wall polypropylene multiplate 96-well unskirted PCR plates (BioRad) sealed with microseal 'B' adhesive (Bio- Rad). Reactions were performed in a BioRad MiniOpticon real time PCR detection system and included an initial denaturation at 95°C for 3 min, 40 cycles of 95°C 30 sec- onds, 58°C 30 sec, 72°C 6 min (with a signal read at the end of each cycle), and a final melting curve to check fidel- ity from 60 – 95°C, with a signal read every 1°C. Gene- specific 20 bp primers for each gene were designed using Primer3 [148] specified to amplify a ~120 bp fragment (+/ - 10 bp) in the last kilobase of the 3' end of the open read- ing frame and coinciding with the region recognised by the corresponding array oligonucleotide. Primer pairs were validated in silico using Amplify [149] to minimise the probability of mispriming or formation of primer dimers and secondary structure. Antibody production and Western blot analysis TbδAd antisera was generated against an expressed gene fragment (residues 222–508) amplified from T. brucei genomic DNA using Herculase DNA polymerase (Strata- gene) with the following primers: 5'-GGAATTCCCAGCT- TCTTAGGTCTAGCGGT-3' and 5'- CCGCTCGAGCTCAACAATAGTTCGCATGTGC-3'. The 865 bp PCR product was subcloned into pPCR-Script, excised with EcoRI, and inserted into pGEX-2TK. Polyclo- nal rabbit antibodies were raised against TbδAd-GST recombinant fusion protein, which was SDS-PAGE-puri- fied due to insolubility when expressed in E. coli, mixed with RIBI adjuvant (Sigma) and used to immunize rabbits on four immunisations spread over a period of five months. 0.5 mg recombinant protein was used per immu- nisation course. For Western blotting, 1 × 107 trypanosome cells were har- vested by centrifuging at 800 g, 10 min, 4°C, washed in phosphate-buffered saline (PBS) and resuspended in 37.5 μl PBS and 12.5 μl 4× Laemmli SDS loading buffer [143]. Lysates were denatured at 95°C for 10 min and analysed by 12.5% SDS-PAGE [143]. Equivalence of protein load- ing was verified by Coomassie Blue staining of a duplicate gel. Proteins were electrophoretically transferred to Immobilon-P membrane (Millipore) using a wet transfer tank (Hoefer Instruments). Nonspecific binding was blocked for 1 hr with Tris-buffered saline, pH 7.4, 0.2% Tween (TBST) supplemented with 5% freeze-dried milk. The membrane was then incubated for 1 hr with primary antibody, also diluted in TBST-milk. Rabbit polyclonal antibody against trypanosome BiP (a kind gift of James Bangs, University of Wisconsin) or the trypanosome clathrin heavy chain [16] were used at 1:1,000 dilution, and rabbit anti-TbδAd (see above) at 1:1,000. After wash- ing 3 times for 5 min in TBST, a commercial secondary anti-IgG rabbit horse-radish-peroxidase conjugate (Sigma) was used at 1:10,000 in TBST for 1 hr, and bound-antibodies were detected on Biomax MR-1 films (Kodak) using H2O2-activated Luminol (Sigma) in 100 mM Tris pH 8.5. Authors' contributions VLK and MCF designed the microarray and the experi- mental approach, VLK performed the microarray analysis and data interpretation, TS generated and validated the VSG RNAi line and SKAN performed immunochemical analysis of the Rab5 overexpressor lines. VLK and MCF drafted the manuscript and figures; all authors read and approved the final manuscript. Additional material Additional file 1 Absence of clustering of membrane-trafficking genes in the T. brucei genome. Panel A: The number of genes identified on each chromosome predicted to have a role in membrane traffic based on sequence similarity and/or domain architecture are plotted against chromosome size (in Mb). Multicopy genes are represented here as a single gene. Panel B: Raw data on which plot A is based. Approximate chromosome sizes are from GeneDB. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-9-298-S1.pdf] Page 23 of 28 (page number not for citation purposes)