BMC Genomics 2009, 10:442 http://www.biomedcentral.com/1471-2164/10/442 scanned (GeneChip Scanner 3000 7G). Cell intensity val- ues and probe detection calls were computed from the raw array data using the Affymetrix GeneChip Operating Soft- ware (GCOS). Further data processing was performed in the R computing environment [40] using packages from the BioConductor software project [41]. Robust Multi- Array Average (RMA) normalisation was applied [42]. Normalised data were then filtered based on the Affyme- trix detection call, so that only probes that had a Present call in at least one of the arrays were retained [43]. Probes with low intensity values (less than 100) in all arrays were also excluded from statistical analysis. Data were then imported into the MultiExperiment Viewer (MeV) soft- ware [44], and statistical analysis was performed with the SAM (Significance Analysis of Microarrays) module [45], implemented as in [46]. A False Discovery Rate (FDR) of 3% was applied to detect significantly differentially expressed genes in DBA patients versus healthy controls. Functional analysis of differentially expressed genes was performed through the use of Ingenuity Pathways Analy- sis [19]. Microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) database with Acces- sion Number GSE14335. Validation of data by real time RT-PCR Genes to be validated were selected on the basis of poten- tial interest and as representative of a wide range of expression fold changes in patients relative to controls. 500 ng of total RNA was reverse transcribed to cDNA using the High Capacity cDNA Archive Kit (Applied Bio- systems) and random primers. Quantitative PCR was per- formed with an AbiPrism7000 instrument (Applied Biosystems) and 1 μl of cDNA was used in a 25-μl final volume reaction containing Power SYBR® Green PCR Mas- ter Mix (Applied Biosystems) and specific primers (see additional file 4: Primers used in the expression analysis by qRT-PCR). cDNA from each sample was examined in triplicate in each experiment. Experimental Ct values were normalised to beta actin, used as endogenous control. Gene expression was calculated in each sample relative to the mean of controls, using the formula 2-ddCt, where dCt is Ctgene-Ctendo and ddCt is dCtsample- mean dCtcontrols. Dif- ferences in gene expression of patients relative to controls were statistically evaluated by t test for independent sam- ples. Mutation analysis DBA patient 3 was further analyzed for exon 2 skipping in RPS19 transcript. The coding sequence spanning exon 1 and exon 4 was PCR-amplified by standard procedures. PCR products were excised from agarose gel, purified with HiYield Gel/PCR DNA Fragments Extraction Kit (Real Bio- tech, Banqiao City, Taiwan) and sequenced in both direc- tions using a Big Dye Terminator® v1.1 cycle sequencing kit (Applied Biosystems) and an Abi PRISM® 3100 genetic analyzer (Applied Biosystems). Primer sequences are available upon request. Authors' contributions FA performed qRT-PCR analysis, gene clustering analysis, mutation analysis and drafted the manuscript. PR per- formed bioinformatic and statistical microarray data anal- ysis, participated in the interpretation of the data and critically revised the manuscript. NC and CC isolated and cultured fibroblasts. HK carried out microarray hybridisa- tions. EG and MFC genotyped the patients and carried out RNA isolation. MA participated in gene clustering analy- sis. UR was involved in diagnosis and follow-up of patients, provided their samples and clinical information. SG, CS and ID contributed to the conception and design of the study, interpretation of the data and critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript. Additional material Additional file 1 Genes differentially expressed in DBA patients identified by microar- ray profiling. The table reports the probeset IDs which are differentially expressed in DBA patients relative to controls, with an FDR of 3%. The gene annotation, chromosome location and fold change of expression in patients relative to controls is also reported. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-442-S1.pdf] Additional file 2 Differentially expressed genes in females relative to males. The table reports the probeset IDs which are differentially expressed in females rel- ative to males, with an FDR of 10%. The gene annotation, chromosome location and fold change of expression in females relative to males is also reported. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-442-S2.pdf] Additional file 3 Differentially expressed genes in female patients relative to male patients. The table reports the probeset IDs which are differentially expressed in female DBA patients relative to male DBA patients, with an FDR of 10%. The gene annotation, chromosome location and fold change of expression in females relative to males is also reported. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-442-S3.pdf] Additional file 4 Primers used in the expression analysis by qRT-PCR. The table reports the sequence of both forward and reverse primers used to amplify genes in the validation experiments by qRT-PCR. The final concentration used in the reactions is also indicated for each primer. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-442-S4.pdf] Page 10 of 12 (page number not for citation purposes)