|
| Status |
Public on May 13, 2011 |
| Title |
MCF-7 +ER siRNA + vehicle rep1 (MD37) |
| Sample type |
RNA |
| |
|
| Source name |
MCF-7cells treated with vehicle and ER siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7 breast cancer cell line
sirna: ER siRNA
agent: control (vehicle)
dose: n/a
|
| Treatment protocol |
Cells were plated at 20% confluence in low glucose phenol red free medium supplemented with 5% charcoal stripped FBS and glutamine 24h-48h prior to transfection. Treatment with vehicle, OH-Tam (100nM), or OH-Tam (500nM) was begun an additional 24h later. Cells were transfected with control siRNA, ERα siRNA or RARα siRNA in 24 well microplates (2.5pmol/well) or 25cm2 flasks (31.25pmol per flask) using 2μl and 12.5 μl of Dharmafect 1 (Thermo Scientific Dharmacon Inc., Lafayette, CO), respectively, according to the vendor’s protocol. The cell culture medium was not replenished for the duration of the experiment.
|
| Growth protocol |
Cells were routinely cultured at 37°C and in 5% CO2 in DMEM supplemented with FBS (10%), penicillin (100unit/ml), streptomycin (100μg/ml) and L-glutamine (2mM). Hormone depleted cells were grown in low glucose phenol-red free media supplemented with 5% charcoal-stripped FBS (v/v) and L-glutamine (2mM) for 48 hours before the experiments
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using the RNeasy Mini kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Isolated total RNA was processed as recommended by Affymetrix, Inc. (Santa Clara, CA). In brief, cDNA was synthesized from the total RNA using the Super-Script Double Stranded cDNA Synthesis kit and T7 Oligo (dT) primers. Using the double stranded cDNA as template, biotin labeled cRNA was generated by in vitro transcription (MEGAscript Labeling Kit) reaction.
|
| |
|
| Hybridization protocol |
The cRNA was fragmented to 35-200 bases length using Affymetrix protocols and hybridized to HG U133 Plus 2.0 array set at 45ºC for 16 hours in an Affymetrix GeneChip® Hybridization Oven 640.
|
| Scan protocol |
Each GeneChip was then washed and stained with Streptavidin–Phycoerythrin conjugate (SAPE; Invitrogen Corp.) using an Affymetrix Fluidics Station 450 and scanned on a Affymetrix GeneChip scanner.
|
| Description |
Gene expression data from MCF-7cells treated with vehicle and ER siRNA
MD37
|
| Data processing |
Scanned image files were analyzed using the Gene Chip Operating System Version 1.4 software, and standard thresholding and filtering operations were used. The data was normalized using house keeping genes. Normalization assumes that for a subset of genes (i.e., housekeeping genes) the ratio of measured expression averaged over the set should be one.
|
| |
|
| Submission date |
Dec 23, 2010 |
| Last update date |
May 13, 2011 |
| Contact name |
Robert J Trumbly |
| E-mail(s) |
[email protected]
|
| Phone |
419-383-4347
|
| Organization name |
University of Toledo
|
| Department |
Biochemistry and Cancer Biology
|
| Street address |
3000 Arlington Avenue
|
| City |
Toledo |
| State/province |
OH |
| ZIP/Postal code |
43614 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE26298 |
Under conditions of hormonal adjuvant treatment the estrogen receptor apoprotein supports breast cancer cell cycling through the retinoic acid receptor α1 apoprotein |
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