|
| Status |
Public on Aug 28, 2020 |
| Title |
ROSE RAS transformed PI3K control |
| Sample type |
RNA |
| |
|
| Source name |
rat ovarian surface epithelium RAS transformed PI3K control
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue/cell type: ovarian surface epithelium cells
genotype/variation: RAS transformed; PI3K control
treatment: LY294007 treated control
|
| Treatment protocol |
The HRASG12V-transformed human embryonal kidney cells (HA1ER) and their immortalized origin cell line HA1EB were described by [Hahn et al. Nature 400, 464-468 (1999)] . They were cultivated in Minimum Essential Medium Eagle, Alpha Modification (MEM Alpha) from Sigma, supplemented with 10% IFS, 2 mM ultraglutamine, 1% penicillin/streptomycin, 0,1mg/ml hygromycin and 0,5µg/ml puromycin. G418 was freshly added to a final concentration of 0,4 mg/ml . ROSE199, rat ovary epithelial cell line and their KRAS transformed derivative cell line ROSEA25 were described in [Tchernitsa et al. Oncogene 23, 4536-4555 (2004)]. Inducible RAS studies were done in HEK cell line transfected with a constuct bearing Ha-RAS under ER promoter. Cells were treated with tamoxifen before RNA was extracted. U0126 MEK inhibitor was dissolved in DMSO and used in 10µM final concentration for 48h before RNA was extracted. LY294002 PI3K inhibitor was dissolved in ethanol and used at final concentration of 50µM.
|
| Growth protocol |
RAS-ROSE abd ROSE cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10 % fetal calf serum (FCS) (Sigma-Aldrich), 2 mM Ultraglutamine 1 (Lonza, BioWhittaker), and 100 units/ml penicillin/streptomycin (Biochrom AG) (standard medium). The medium was supplemented with G418 (400 μg/μl). 45000 RAS-ROSE cells were seeded into 6-well plates (BD Falcon) in standard medium 24 h prior to transient transfection of siRNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from cell lines using the mirVanaTM miRNA Isolation Kit from Ambion® (Austin, TX, USA) according to the manufacturer’s instructions. In order to avoid cross-labeling of premature miRNA we used the gel purification option. Subsequently the quality of the small RNA fraction was controlled on a 15% PAAG gel stained with ethidium bromide.
|
| Label |
biotin
|
| Label protocol |
Standard Affymetrix labeling protocol
|
| |
|
| Hybridization protocol |
Standard Affymetrix hybridization protocol
|
| Scan protocol |
Standard Affymetrix scanning protocol
|
| Description |
replicate 1
|
| Data processing |
The data were normalised using dchip (Build date April 9 2009) normalized
|
| |
|
| Submission date |
May 09, 2013 |
| Last update date |
Aug 28, 2020 |
| Contact name |
Wasco Wruck |
| E-mail(s) |
wasco.wruck@med.uni-duesseldorf.de
|
| Organization name |
Universitätsklinikum Düsseldorf
|
| Street address |
Moorenstr. 5
|
| City |
Düsseldorf |
| ZIP/Postal code |
40225 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1355 |
| Series (2) |
| GSE46301 |
A conserved microRNA signature related to cellular transformation by the RAS oncogene (Affymetrix mRNA) |
| GSE46806 |
A conserved microRNA signature related to cellular transformation by the RAS oncogene |
|
