Transcriptome and tissue expression analysis.

Total RNA from early-passage rheumatoid synovial fibroblasts (3 early-passage lines per experimental group) was isolated using RNeasy miniprep columns (Qiagen), and labeling and hybridization were performed with the Genisphere Array 900 Expression Array Detection Kit according to the manufacturer’s protocol. The OMM25K oligonucleotide gene array set (18,000 genes) was used (http://support.illumina.com/array/array_kits/humanht-12_v4_expression_beadchip_kit.html) and the cDNA probe and the fluorescent 3DNA reagent were hybridized to the microarray in succession using an Advalytix Slide Booster hybridization station. The hybridized slides were scanned with a Gene-Pix 4000 scanner (Axon Instruments), and raw data were analyzed with GenePix 5.0 analysis software. Hierarchical clustering analysis was performed using GeneSpringGX 7.3 (Agilent Technologies) to reveal relationships among the different experimental conditions. Pearson correlation was used to measure the similarity of the expression levels. Responsive genes were extracted from an unsupervised, full transcriptional profile analysis by a combination of statistical testing of absolute and relative changes in expression across the different experimental conditions and a permutation-based test to estimate false discovery. Statistical analysis among experimental groups was performed using the Student’s t test, with a FDR of less than 0.05 and a fold change of more than 1.5 considered as differential expression. Original data were deposited at the International MIF Consortium website (http://imc.isd-muc.de/) under filename MIF:ICBP90 Study.

A previously collected human synovial tissue registry, comprising samples obtained in accord with an IRB-approved protocol for subjects undergoing arthroplasty and/or synovectomy and fulfilling the American College of Rheumatology criteria for disease (57, 58), was accessed for comparative mRNA expression analysis. Synovial samples were processed for expression analysis on GeneChip Human Genome U133 Plus 2 Affymetrix arrays, and the accessed data are in GEO data sets {"type":"entrez-geo","attrs":{"text":"GSE48780","term_id":"48780"}}GSE48780 and {"type":"entrez-geo","attrs":{"text":"GSE7669","term_id":"7669"}}GSE7669.