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[Analysis of gene expression patterns in rheumatoid synovial fibroblasts using RAP-PCR for differential display]
U Müller-Ladner1, M Judex, H P Jüsten
1Klinik und Poliklinik für Innere Medizin I, Universität Regensburg. ulf.mueller-ladner@klinik.uni-regensburg.de
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) using RAP-PCR for differential display.
Methods:
After extraction of total RNA, the first step of RAP-PCR was performed using various different arbitrary 10-12-base primers for first-strand cDNA synthesis. Second-strand synthesis was achieved by cycling at low stringency conditions for 35 cycles using different arbitrary 10-base primers, followed by electrophoretic separation and sequence analysis of the amplified fingerprint products.
Results:
On average, approximately 70 different RNAs were obtained per primer, of which most were expressed both by RA and OA synovial fibroblasts. Using 26 different primer combinations, in total 12 cDNAs were differentially expressed between RA and OA synovial fibroblasts. In the RA group strong amplification of distinct PCR products suitable for sequencing could be observed. 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 identify differentially expressed genes in rheumatoid synovial fibroblasts potentially involved in the specific pathophysiology of RA.
Insights
Researchers identified genes involved in cell cycle and metabolism that are differentially expressed in rheumatoid arthritis (RA) synovial fibroblasts compared to osteoarthritis (OA) fibroblasts using RNA arbitrarily primed PCR (RAP-PCR). This method helps uncover genes contributing to RA pathogenesis.
Area of Science:
- Molecular Biology
- Rheumatology
Context:
- Rheumatoid arthritis (RA) involves synovial fibroblast invasion, leading to cartilage and bone destruction.
- The specific genetic mechanisms driving this aggressive RA phenotype are not fully understood.
Purpose:
- To identify genes differentially expressed in RA synovial fibroblasts compared to osteoarthritis (OA) synovial fibroblasts.
- To utilize RNA arbitrarily primed PCR (RAP-RAP-PCR) for differential gene expression analysis.
Summary:
- RNA arbitrarily primed PCR (RAP-PCR) was employed to analyze gene expression in synovial fibroblasts from RA and OA patients.
- Out of approximately 70 RNAs per primer, 12 cDNAs showed differential expression between RA and OA fibroblasts.
- Sequencing identified these differentially expressed genes as being involved in cell cycle regulation and metabolism.
Impact:
- RNA arbitrarily primed PCR (RAP-PCR) proves effective for identifying genes implicated in RA pathophysiology.
- The findings provide insights into the molecular basis of RA-associated synovial fibroblast aggression.