|
| Status |
Public on Aug 28, 2020 |
| Title |
ratA25LY_ratA25Eth_r2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ratA25_Eth_48
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: KRAS transformed ROSEA25
tissue/cell type: ovarium surface epithelium cells
treatment: treated with ethanol for 48h
|
| Treatment protocol |
Induble RAS studies were done in HEK cell lines transfected with a constuct bearing Ha-RAS under ER promoter (33) Cells were treated with …µM of 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 |
The HRASG12V-transformed human embryonal kidney cells (HA1ER) and their immortalized origin cell line HA1EB were described by Hahn et al. (32) . 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 decribed in Tchernitsa, 2000.
|
| 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 maufacturer’s instructions. In order to avoid cross-labeling of premature miRNA we used the gel purification option. Sunseqneutly the quality of the small RNA fraction was controlled on a 15% PAAG gel stained with ethidium bromide.
|
| Label |
Alexa3
|
| Label protocol |
100 ng of small RNA fraction were labelled using the NCODETM miRNA labeling system (Invitrogen, CA, USA) according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
ratA25_LY_48
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: KRAS transformed ROSEA25
tissue/cell type: ovarium surface epithelium cells
treatment: treated with LY294002 for 48h
|
| Treatment protocol |
Induble RAS studies were done in HEK cell lines transfected with a constuct bearing Ha-RAS under ER promoter (33) Cells were treated with …µM of 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 |
The HRASG12V-transformed human embryonal kidney cells (HA1ER) and their immortalized origin cell line HA1EB were described by Hahn et al. (32) . 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 decribed in Tchernitsa, 2000.
|
| 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 maufacturer’s instructions. In order to avoid cross-labeling of premature miRNA we used the gel purification option. Sunseqneutly the quality of the small RNA fraction was controlled on a 15% PAAG gel stained with ethidium bromide.
|
| Label |
Alexa3
|
| Label protocol |
100 ng of small RNA fraction were labelled using the NCODETM miRNA labeling system (Invitrogen, CA, USA) according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Microarray hybridisation and washing was performed according to the same manual on Slide BoosterTM hybridisation station (Advalytix, Germany).
|
| Scan protocol |
Microarray images were obtained on an Agilent G2565AA scanner at 10 µm resolution.
|
| Description |
always two replica with a dye swap
|
| Data processing |
Image analysis was carried out using the ImaGene software (BioDiscovery, Inc., El Segundo, USA). For each target miRNA, two independent hybridizations were done by inverting the Alexa3 and Alexa5 fluorochromes in the labelling reactions. Background subtraction and normalisation was done with GeneSite software (BioDiscovery, Inc, El Segundo, USA) by global scaling normalization, the median to set the average to 1 for all arrays.. For statistical analysis BRB-ArrayTools software, developed by Dr. Richard Simon and Development Team was used
|
| |
|
| 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 |
GPL10352 |
| Series (2) |
| GSE46794 |
A conserved microRNA signature related to cellular transformation by the RAS oncogene (miRNA) |
| GSE46806 |
A conserved microRNA signature related to cellular transformation by the RAS oncogene |
|
