|
| Status |
Public on Dec 20, 2014 |
| Title |
C6cells_AngII+PD6h_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
C6 glioma cells, Angiotensin II + PD123319 (AT2 antagonist), 6 hours, replicate 4
|
| Organism |
Rattus norvegicus |
| Characteristics |
ang ii treatment (10-7m): Ang II
ang ii receptors antagonists (10-5m): PD123319 (AT2 antagonist)
time interval: 6 hours
|
| Treatment protocol |
C6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 Units/ml penicillin and 100 µg/ml streptomycin. Cells were seeded in cell culture dishes and incubated at 37°C/ 5% CO2 until becoming confluent. Then, these cells were pre-treated (30 minutes) with either AT1 receptor antagonist (Losartan: 10-5M) or AT2 receptor antagonist (PD123319: 10-5M) followed by Ang II treatment (10-7 M) according to the treatment scheme: Group 1 – control; Group 2 – cells only treated with Ang II; Group 3 – cells pre-treated (30 minutes) with Losartan and then treated with Ang II; Group 4 – cells pre-treated (30 minutes) with PD123319 and then treated with Ang II.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from samples at 3 and 6 hours intervals using Trizol reagent (Life Technologies, USA) and purified using RNeasy Spin Columns (Qiagen, USA). RNA quantity was determined using a Nanovue spectrophotometer (GE Healthcare, USA). The RNA quality was performed using a 2100 Bioanalyzer with an RNA 6000 Nano kit and Ladder (Agilent Technologies, USA), according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Microarray-Based Gene Expression Analysis - Quick Amp Labeling (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen). RNA concentration and purity was determined by reading the absorbance at 260 and 280 nm on a spectrophotometer (NanoVue, GE Health Care, USA).
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes and hybridized to Agilent Whole Rat Genome Oligo Microarrays (G2519F-028282) for 17 hours at 65°C in a rotating Agilent hybridization oven.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Bundle using one color scan setting for 1x44k array slides.
|
| Description |
Gene expression of C6 glioma cells submitted to Ang II and PD123319 treatment at 6 hours interval
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software version 9.5.3.1 (Agilent) using default parameters (protocol GE1-v5_95 Feb07 and Grid: 028282_D_F_20100528) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
May 30, 2013 |
| Last update date |
Dec 20, 2014 |
| Contact name |
Hatylas Azevedo |
| Organization name |
University of São Paulo
|
| Department |
Pediatrics
|
| Street address |
Doutor Arnaldo Avenue, 455
|
| City |
São Paulo |
| State/province |
Sao Paulo |
| ZIP/Postal code |
01246903 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL14746 |
| Series (1) |
| GSE47529 |
Transcriptional network analysis reveals that AT1 and AT2 Angiotensin II receptors are both involved in the regulation of genes essential for glioma progression |
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