|
| Status |
Public on Oct 01, 2010 |
| Title |
Human_osteoblast_sample_#593rep3_DEX_treated_24h |
| Sample type |
RNA |
| |
|
| Source name |
Primary human osteoblasts, DEX, 24h
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: trabecular bone
cell type: osteoblasts
condition: dexamethasone
timepoint: 24h
gender: male
|
| Treatment protocol |
At 70-80% confluence, the cells were passaged and sub-cultured in 6-well plates (100,000 cells/well) for 12 days. The culture medium was changed twice weekly. 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 0.1 µg/ml of rhBMP-2 (bone morphogenetic protein 2), 100 nM of DEX (dexamethasone), 100 nM of IGF-1 (insulin-like growth factor 1), 1µM of PGE2 (prostaglandin E2), 100 nM of PTH (parathyroid hormone) (1-34), 0.1 nM of TNF-α (tumor necrosis factor alpha) 100 nM of 1.25 VitD3 (vitamin D3) and with the same concentration of vehicle, respectively. At the two time points, the cell medium was removed and the cells were harvested by adding 600µL 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.
|
| Growth protocol |
The cells were grown in medium containing α-MEM supplemented with 2 mmol/l L-Glutamine, 100U/mL penicillin, 100mg/mL streptomycin, and 10% fetal bovine serum at 37°C with 5% CO2. The culture medium was changed twice weekly.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cell lysates resuspended in 600µl RLT lysis buffer using the RNeasy® Mini Kit. High RNA quality was confirmed for all samples using the Agilent 2100 BioAnalyzer and the concentrations were determined using Nanodrop ND-1000. The PTH-treated samples failed in the RNA isolation experiment and thus were not included in the subsequent expression profiling experiment.
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays.
|
| |
|
| Hybridization protocol |
Equivalent amount of cRNA (750ng) was hybridized on each array according to the standard Illumina hybridization protocol.
|
| Scan protocol |
The BeadArrays were scanned according to the standard Illumina scanning protocol using the Illumina®BeadArray Reader (version 1.7.0.44) and the BeadScan software (version 3.5.31.17122).
|
| Description |
HOb_593:3_DEX_24h
|
| Data processing |
The images were analyzed in Illumina®BeadStudio 3 application and raw data were outputted in txt files. The raw data was then imported to BioConductor in R 2.5.0 using the lumi package for variance-stabilizing transformation and robust spline normalization.
|
| |
|
| Submission date |
Apr 20, 2010 |
| Last update date |
Aug 31, 2010 |
| Contact name |
Elin Grundberg |
| Organization name |
McGill University
|
| Street address |
740 Dr Penfield Avenue
|
| City |
Montreal |
| ZIP/Postal code |
H3A1A4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6104 |
| Series (2) |
| GSE21725 |
Global analysis of the impact of environmental perturbation on cis-regulation of gene expression: DEX, 24hr |
| GSE21728 |
Global analysis of the impact of environmental perturbation on cis-regulation of gene expression |
|
