|
| Status |
Public on Jul 18, 2009 |
| Title |
10-1 cells empty vector |
| Sample type |
RNA |
| |
|
| Source name |
10-1 cells, empty vector
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 10-1
genotype: p53 null
|
| Treatment protocol |
Cells were cultured in complete medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum) maintained at 37°C in a humidified atmosphere of 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from cells using TRIzol® (Invitrogen) and digested with RNase-free DNase I (Qiagen) according to the manufacturer’s instructions. The quality and integrity of the total RNA was evaluated with the Agilent 2100 Bioanalyzer and the concentration was measured using a NanoDrop spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x44k array slides.
|
| Description |
BALB/c mouse embryo fibroblasts
Gene expression in MEFs lacking p53 and stably transfected with the empty vector
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_10_Apr08 and Grid: 014868_D_F_20080627) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jul 17, 2009 |
| Last update date |
Jul 17, 2009 |
| Contact name |
Genrich Tolstonog |
| E-mail(s) |
genrich.tolstonog@hpi.uni-hamburg.de
|
| Phone |
+49-40-48051235
|
| Fax |
+49-40-48051117
|
| Organization name |
Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology
|
| Department |
Department of Tumor Virology
|
| Street address |
P.O. Box 20 16 52
|
| City |
Hamburg |
| ZIP/Postal code |
D-20206 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE17178 |
Wild-type p53 enhances efficiency of simian virus 40 large-T-antigen-induced cellular transformation |
|
