|
| Status |
Public on Mar 01, 2015 |
| Title |
SiO2NPs_0.1µg/cm²_rep biol3_rep tech3 [US11183901_252665213950_S01_GE1_1010_Sep10_1_3] |
| Sample type |
RNA |
| |
|
| Source name |
A549 exposed to 0.1µg/cm² SiO2NPs, 24h
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Lung
cell type: Alveolar epithelium
cell line: A549
|
| Treatment protocol |
For microarray experiments, the A549 cells were seeded in 6-well plates to collect sufficient amounts of RNA. Three different cultures were exposed to five concentrations of SiO2 NPs (0.1, 1.0, 1.5, 3.0 and 6.0 µg/cm²) and serum-free medium (control cells) for 24h, and exposed to two concentrations of SiO2 NPs (3.0 and 6.0 µg/cm²) for 72h. Each condition of exposure to NPs was compared with control cells.
|
| Growth protocol |
A549 cells (ATCC, Manassas, VA, US) were cultured in DMEM/F12 medium supplemented with 10% FCS (LGC Standards, Middlesex, UK) and penicillin/streptomycin (100 U/mL, 100 µg/mL) in a humidified incubator at 37 °C, and 5% CO2. Cells were used between passages 20 to 40. Cells were passed twice a week and the medium was changed three times per week, keeping confluence below 80%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using the RNeasy Mini Kit (Qiagen). RNAs were quantified with the NanoDrop 1000 spectrophotometer and their qualities were assessed with the Agilent 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
RNA samples were amplified and labeled with cyanine-3 fluorophore using a Low Input Quick Amp Labeling Kit (Agilent), according to the supplier’s protocol. The efficiency of fluorescent labelling was controlled by UV spectroscopy (Nanodrop 1000).
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| |
|
| Hybridization protocol |
1.65 µg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 µL of 2X Gex Hybridization Buffer Hi-RPM was added to the fragmentation mixture and hybridized to Human V2 4x44K oligo microarrays (Agilent) 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 at ambiant atmosphere.
|
| Scan protocol |
Microarrays were scanned immediately after washing on the Agilent Scanner using one color scan setting.
|
| Description |
Gene expression after 24h exposure to NPs
Microarray #: 252665213950
Block: 3
|
| Data processing |
The scanned images were analyzed with Feature Extraction software (Agilent technologies) using one-color treatment to obtain background subtracted and spatially detrended Processed Signal intensities. Non-uniform outliers were excluded. Data were statistically analyzed with Genespring GX11 software (Agilent technologies).
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| |
|
| Submission date |
Dec 03, 2014 |
| Last update date |
Mar 01, 2015 |
| Contact name |
Odette Prat |
| E-mail(s) |
odette.prat@cea.fr
|
| Organization name |
CEA
|
| Department |
Life Science
|
| Lab |
SBTN
|
| Street address |
Site de Marcoule
|
| City |
Bagnols sur Cèze |
| ZIP/Postal code |
30207 |
| Country |
France |
| |
|
| Platform ID |
GPL10332 |
| Series (1) |
| GSE63806 |
Dose dependent toxicological response of lung cells exposed to silica nanoparticles |
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