cell line: RWPE-1 cell type: Immortalized normal prostate epithelial cells. treatment: 100 nM 1,25(OH)2D time point: 48 hours
Treatment protocol
RWPE1 cells were treated with medium containing 100 nM of 1,25(OH)2D or vehicle (0.1% ethanol) for 6, 24 or 48 hours (n=4 per treatment, 24 total samples).
Growth protocol
RWPE1 cells were plated in T75 flasks (1x10^6 cells per flask) and grown until cells reached 60% confluence. For the 48 hours time point media was replaced at 24 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the cells using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) in accordance with the manufacturer’s instructions. Isolated total RNA was further purified using the RNeasy kit (Qiagen, Valencia, CA). The quality of the isolated RNA was confirmed using agarose gel electrophoresis.
Label
biotin
Label protocol
The transcripts levels in each sample were measured by using the Affymetrix HU133 plus 2.0 GeneChip (Affymetrix, Santa Clara, CA; 54,210 probe sets covering over 47,000 transcripts and splice variants). RNA labeling was carried out by using standard Affymetrix protocols (Affymetrix, Santa Clara, CA).
Hybridization protocol
Chip hybridization was carried by using standard Affymetrix protocols (Affymetrix, Santa Clara, CA) at the Ohio State University Comprehensive Cancer Center (Columbus, OH).
Scan protocol
Chip scanning was carried out by using standard Affymetrix protocols (Affymetrix, Santa Clara, CA) at the Ohio State University Comprehensive Cancer Center (Columbus, OH).
Description
Gene expression of 1,25(OH)2D treated RWPE1 at 48h.
Data processing
Microarray data from CEL files for all 24 chips was normalized simultaneously and expression levels were generated using the gcRMA package in Bioconductor.
