Dataset features


Application: Gene expression microarray analysis
Number of samples: 60
Release date: Jul 20 2009
Last update date: Oct 29 2018
Access: Public
Diseases: Arthritis, Rheumatoid, Rheumatic Diseases
Dataset link Adapted Boolean Network Models for Extracellular Matrix Formation

Experimental Protocol

Patients and tissue samples: Synovial membrane samples were obtained within 10 min following tissue excision upon joint replacement/synovectomy from RA and OA patients (n = 3 each). After removal, tissue samples were frozen and stored at -70°C. Informed patient consent was acquired and the study was approved by the ethics committees of the respective universities. RA patients were classified according to the American College of Rheumatology (ACR) criteria, OA patients according to the respective criteria for osteoarthritis. The preparation of primary semi-transformed synovial fibroblasts from RA and OA patients was performed as previously described (Zimmermann et al., Arthritis Res. 2001;3(1):72-6). Briefly, the tissue samples were minced and digested with trypsin/collagenase P. The resulting single cell suspension was cultured for seven days. Non-adherent cells were removed by medium exchange. SFB were then negatively purified using Dynabeads® M 450 CD14 and subsequently cultured over 4 passages in DMEM containing 100 μg/ml gentamycin, 100 μg/ml penicillin/streptomycin, 20 mM HEPES, and 10% FCS. Cell stimulation and isolation of total RNA : At the end of the fourth passage, the SFB were stimulated by 10 ng/ml TGFβ or TNFα in serum-free DMEM for 0, 1, 2, 4, and 12 h. At the end of each time point, medium was removed and the cells were digested with trypsin/versene (0.25%). Following centrifugation and washing with PBS, the cells were lysed with RLT buffer (Qiagen) and frozen at 70°C. Total RNA was isolated using the RNeasy Kit (Qiagen) according to the supplier's recommendation. Microarray data analysis: RNA probes were labelled according to the supplier's instructions (Affymetrix®, Santa Clara, CA, USA). Analysis of gene expression was performed using U133 Plus 2.0 RNA microarrays. Hybridization and washing was performed according to the supplier's instructions. Microarrays were analyzed by laser scanning (Hewlett-Packard Gene Scanner). Background-corrected signal intensities were determined using the MAS 5.0 software (Affymetrix®) and normalized among arrays to facilitate comparisons between different patients. For this purpose, arrays were grouped according to patient class the respective stimulus (TGFβ and TNFα, n=6 each). The arrays in each group were normalized using quantile normalization. See publication for further details.










René Huber

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