|
| Status |
Public on May 21, 2012 |
| Title |
rapamycin 12h treatment, replicate 3 polysome-associated RNA |
| Sample type |
RNA |
| |
|
| Source name |
rapamycin 12h treatment, replicate 3 polysome-associated RNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
tumor type: Breast cancer
rna source: polysome-associated
treatment: rapamycin
experiment: 3
|
| Treatment protocol |
Cells were treated with vehicle, rapamycin (100nM), PP242 (1M) and metformin (10mM) for 12h
|
| Growth protocol |
MCF7 cells were obtained from ATCC and grown in RPMI (Invitrogen), supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin (all from Invitrogen) at 37oC and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Polysome profile analysis was carried out as previously described (Dowling RJ, Zakikhani M, Fantus IG, Pollak M, & Sonenberg N (2007) Cancer Res 67, 10804-10812). A parallel sample was collected from post-nuclear lysates that are loaded onto the sucrose gradient and RNA was isolated using Trizol (Invitrogen) (“cytoplasmic mRNA”). Polysome fractions with mRNA associated with >3 ribosomes were pooled (“polysome-associated” mRNA) and RNA was isolated using Trizol.
|
| Label |
biotin
|
| Label protocol |
500 µg cytoplasmic or polysome-associated RNA was used as starting material for the 3’ IVT Express Kit (Affymetrix).
|
| |
|
| Hybridization protocol |
The resulting labelled samples were hybridized to the Human Genome U133 Plus 2.0 gene arrays from Affymetrix according the instructions of the manufacturer
|
| Scan protocol |
Arrays were scanned using the GeneArray Scanner 3000 according to the instructions from the manufacturer.
|
| Description |
MCF7 treated with rapamycin for 12h follwed by extraction of polysome-associated RNA.
|
| Data processing |
Data was extracted and normalized using a custom CDF and Robust Multi-array Average (RMA) in R (r-project.org) with default settings.
|
| |
|
| Submission date |
Mar 27, 2012 |
| Last update date |
May 21, 2012 |
| Contact name |
Ola Larsson |
| E-mail(s) |
ola.larsson@ki.se
|
| Phone |
+46 (0)8 517 73280
|
| Organization name |
Karolinska Institutet
|
| Department |
Department of oncology-pathology
|
| Street address |
CCK, R8:01
|
| City |
Stockholm |
| ZIP/Postal code |
171 76 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL14877 |
| Series (1) |
| GSE36847 |
Distinct perturbation of the translatome by the anti-diabetic drug metformin |
|
