Analysis of gene expression patterns in rheumatoid synovial fibroblasts using RAP-PCR for differential display

Objective: Destruction of articular cartilage and bone by invading synovial fibroblasts is a typical histopathologic feature in rheumatoid arthritis ( RA). However, little is known about specific up- or downregulation of genes leading to this aggressive phenotype. Thus, our aim was to identify genes, which are differentially expressed in RA synovial fibroblasts as compared to synovial fibroblasts derived from patients with osteoarthritis (OA) usin g RAP-PCR for differential display. Methods: After extraction of total RNA, the first step of RAP-PCR was perfo rmed using various different arbitrary 10-12-base primers for first-strand cDNA synthesis. Second-strand synthesis was achieved by cycling at low stri ngency conditions for 35 cycles using different arbitrary 10-base primers, followed by electrophoretic separation and sequence analysis of the amplifi ed fingerprint products. Results: On average, approximately 70 different RNAs were obtained per prim er, of which most were expressed both by RA and OA synovial fibroblasts. Us ing 26 different primer combinations, in total 12 cDNAs were differentially expressed between RA and OA synovial fibroblasts. In the RA group strong a mplification of distinct PCR products suitable for sequencing could be obse rved. Sequence analysis identified these PCR products as highly homologous to various genes involved in regulation of cell cycle and metabolism. Conclusion: The data indicate that RAP-PCR is a suitable method to identity differentially expressed genes in rheumatoid synovial fibroblasts potentia lly involved in the specific pathophysiology of RA.