HOb derived from trabecular bone explants, Gender: Male, Age:64
Extracted molecule
total RNA
Extraction protocol
The cells were harvested by adding 500µL of RLT buffer (Qiagen, GmbH, Germany). The cell lysates were homogenized by using QIAshredder (Qiagen, GmbH, Germany) homogenizers and stored in -70°C until RNA extraction. RNA was isolated from the cell lysates using the RNeasy® Mini Kit (Qiagen, GmbH, Germany). High RNA quality was confirmed for all samples using Agilent 2100 BioAnalyzer (Agilent technologies, Palo Alto, CA, USA) and the concentrations were determined using Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA).
Arrays were scanned with the Affymetrix GeneChip® Scanner 3000 7G and the data was extracted using Microarray Analysis Suite 5.0 (Affymetrix).
Description
Primary human osteoblast like cells (HOb) were cultured in three biological replicates in cell medium containing α-MEM supplemented with 2 mmol/l L-Glutamine, 100U/mL penicillin, 100mg/mL streptomycin, and 10% fetal bovine serum and grown at 37°C with 5% CO2 until confluence. At 70-80% confluence, each replicate was trypsinized and sub-cultured for 12 days. Prior to treatment, the cells were starved for 20h by adding complete cell medium containing 0.5% fetal bovine serum. The cells were then incubated for 2h and 24h with 10-7 M of dexamethasone and 10-4 mg/ml of rhBMP-2 with the same concentration of vehicle, respectively.
Data processing
Expression values were calculated using the Robust Multichip Average method (Irizarry RA, Hobbs B, Collin F, et al. 2003. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4: 249-64)
