cell type: pooled Total RNA of OA Cartilage from five patients (female, mean age 74 years, range 61-85) undergoing knee alloarthroplasty at timepoint 7d
Treatment protocol
P0-cells were cultured in high-density pellet cultures. Briefly, 1,500,000 cells were placed into a conical polypropylene tube in chondrogenic medium. The cells were centrifuged at 500xg for 10 minutes. Cell pellets were cultured in DMEM with 1% ITS (Insulin-Transferrin-Selenium 100x, Invitrogen, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin, 210 µmol ascorbic acid, 10 nmol dexamethasone (Sigma Aldrich, Germany) and 10 ng/ml TGF-ß3 (Strathmann Biotec, Germany) at 37°C, 5% CO2 with medium changes every third day.
Growth protocol
Chondrocytes were isolated by collagenase (Collagenase Type I, Worthington; Cat.No.: CLS1) digestion overnight and expanded in DMEM (DMEM, Low Glucose, 1x, Invitrogen; Cat.No.: 21885-108) supplemented with 10% FCS; 1% Penicillin/Streptomycin.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted after 0, 3, 6, 7 and 14 days using a two-step extraction protocol as described elsewhere [Hoemann et. al, 2002]. Briefly, 1 pellet was mixed with 350 µl of RLT-β-mercapto-ethanol solution (RNeasy kit, Qiagen, Germany) and incubated for 30 minutes at 4°C in a thermomixer (shaking vigorously). After centrifugation at 15,000 x g for 10 minutes at 4°C the procedure was repeated with the pellet and 250 µl of RLT-β-mercapto-ethanol solution and a centrifugation at 21,000 x g (10 minutes). Both supernatants were mixed and the RNeasy procedure was carried out as described by the manufacturer.
Label
Biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol.
Hybridization protocol
Hybridization (16h x 45°C) was processed according to the standard Affymetrix protocol.
Scan protocol
Affymetrix GeneArray Scanner3000
Description
Gene expression data from OA Cartilage or mesenchymal stem cells extracted after different timepoints (0d, 3d, 7d and 14d).
Data processing
The data were analyzed with a commercial software called JMP Genomics, version 3.0, from SAS. Gene expression profiling was performed using arrays of human133_plus2 -type from Affymetrix. A Custom CDF Version 10 with Entrez based gene definitions was used to annotate the arrays. The Raw fluorescence intensity values were normalized applying quantile normalization.
