Clinic of Orthopedics, FSU Jena, Waldkrankenhaus “Rudolf Elle”, Eisenberg, Germany
Treatment protocol
Synovial tissue was obtained from open joint replacement surgery or arthroscopic synovectomy at the Clinic of Orthopedics, Waldkrankenhaus “Rudolf Elle” (Eisenberg, Germany). Patients with RA or OA (n = 6 each for gene expression analysis and further patients for validation experiments) were classified according to the ACR criteria.
Growth protocol
The tissue samples were minced, digested with trypsin/collagenase P, and the resulting single cell suspension cultured for 7 days. Non-adherent cells were removed by medium exchange. SFB were then negatively purified using Dynabeads® M-450 CD14 and subsequently cultured over 2 passages in DMEM containing 100 μg/ml gentamycin, 100 μg/ml penicillin/streptomycin, 20 mM HEPES and 10% fetal calf serum (FCS, all from PAA Laboratories, Cölbe, Germany).
Extracted molecule
total RNA
Extraction protocol
At the end of the 2nd passage, the SFB were starved with medium containing 1% fetal calf serum (FCS) for 72 h, to minimize stimulating effects by serum components. After washing with PBS, the cells were lysed with RLT buffer (Qiagen, Hilden, Germany) and frozen at -70°C. Total RNA was isolated using the RNeasy Kit (Qiagen) according to the supplier’s recommendation.
Label
biotin
Label protocol
RNA probes were labeled according to the supplier’s instructions (Affymetrix, Santa Clara, CA, USA).
Hybridization protocol
Hybridization and washing of gene chips was performed according to the supplier’s instructions (http://www.affymetrix.com).
Scan protocol
Microarrays were analyzed by laser scanning (Hewlett-Packard Gene Scanner).
Description
see publication for details
Data processing
Background-corrected signal intensities were determined using the MAS 5.0 software (Affymetrix).
