|
| Status |
Public on Aug 01, 2012 |
| Title |
RAS-ROSE cells treated with Otx1 siRNA 1 |
| Sample type |
RNA |
| |
|
| Source name |
Otx1_siRNA1
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: RAS-ROSE: KRAS-transformed Rose 199 cells
treatment: with Otx1 siRNA 1
|
| Treatment protocol |
Cells were transfected twice with an interval of 24 h using Oligofectamine (Invitrogen), serum-free OptiMEM (Invitrogen) and a final siRNA concentration of 1 nM according to the manufacturer's specifications. After 4 h of incubation, FCS was added to the medium at a final concentration of 10 %.
|
| Growth protocol |
RAS-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 extraction were prepared using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. CDNA synthesis was performed using the Ambion® WT Expression Kit (Ambion).
|
| Label |
biotin
|
| Label protocol |
5.5 μg of the single-strand DNA was enzymatically fragmented with a combination of uracil DNA glucosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) and biotinylated using the WT terminal labeling kit (Affymetrix).
|
| |
|
| Hybridization protocol |
Samples were hybridized accordingly to the manufacturer’s protocol using Hybridization Wash and Stain Kit (Affymetrix). The hybridization at 45°C and 60 rpm for 16hrs, the staining and washing were carried out as recommended by Affymetrix (Affymetrix).
|
| Scan protocol |
GeneChipScanner 3000 G7 system (Affymetrix)
|
| Description |
RAS-ROSE: KRAS-transformed Rose 199 cells (Tchernitsa et al., 2004; Adams and Auersperg, 1985)
RAS-ROSE cells treated with Otx1 siRNA 1
|
| Data processing |
CEL files were analysed using bioconductor oligo package in R, normalisation was done using rma with target probeset.
pd.ragene.1.0.st.v1
pd.ragene.1.0.st.v1
|
| |
|
| Submission date |
Jun 07, 2012 |
| Last update date |
Aug 01, 2012 |
| Contact name |
Nils Blüthgen |
| E-mail(s) |
nils.bluethgen@charite.de
|
| Organization name |
Charite Universitätsmedizin Berlin
|
| Department |
Institute of Pathology
|
| Street address |
Chariteplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10741 |
| Series (2) |
| GSE38584 |
Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach (7TF and control) |
| GSE38614 |
Hierarchical regulation in a KRAS pathway-dependent transcriptional network revealed by a reverse-engineering approach |
|
