|
| Status |
Public on Mar 04, 2014 |
| Title |
AN-48h-NAC |
| Sample type |
RNA |
| |
|
| Source name |
Normal astrocytes, NAC, 48h
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: normal astrocytes
agent: NAC
exposure time: 48h
|
| Treatment protocol |
Human astrocytes were treated with the antioxidant reagent N-Acetyl-L-Cysteine (NAC) and kept in a humidified 5% CO2 atmosphere at 37°C for the indicated time period (48h).
|
| Growth protocol |
Human astrocytes were cultured in human astrocyte medium (ScienCell).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
miRNeasy Mini Kit (Qiagen) with DNase treatment. RNA concentration and purity were determined from measuring absorbance at 260 and 280 nm; 2 μg total RNA was run on a 1% denaturing gel and 100 ng were loaded on the 2100 Bioanalyzer (Agilent, Palo Alto, CA) to verify RNA integrity.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA as detailed in the One-color microarray-based gene expression analysis protocol, v5.5 (www.agilent.com). Dye incorporation and cRNA yield were checked with the NanoDrop.
|
| |
|
| Hybridization protocol |
Labelled cRNAs were hybridized to whole-genome oligonucleotide microarrays (Agilent Technologies) following the manufacturer's instructions (One-color microarray-based gene expression analysis protocol, v5.5). Briefly, 1.5 υg 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 100 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 50 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome 4x44K Oligo Microarrays 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), 1 minute with 37°C GE Wash buffer 2 (Agilent), with the Stabilization solution (Agilent) for 1 minute at room temperature, then dried immediately by rinsing slides into Drying solution (Agilent) for 30 seconds.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G4845A) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm). Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10%. Microarray performance was assessed by QC metric tool.
|
| Description |
Gene expression analysis.
Human astrocytes were obtained from ScienCell (San Diego, CA).
|
| Data processing |
The scanned TIFF images were analyzed with Feature Extraction Software version 10 (Agilent) using default parameters (Agilent protocol GE1-v5_95_Feb07). The output of Feature Extraction was analyzed with the Agi4x44PreProcess package to obtain background subtracted (Agilent parameter gBGSubSignal). Raw data was analyzed using GeneSpring software from Agilent. Normalization of the data was done in GeneSpring using the Quantile normalization.
|
| |
|
| Submission date |
Feb 05, 2013 |
| Last update date |
Mar 05, 2014 |
| Contact name |
Massimiliano Monticone |
| E-mail(s) |
massimiliano.monticone@istge.it
|
| Organization name |
IRCCS AOU San Martino – IST, Genova
|
| Lab |
S.S. Biofisica e Citometria
|
| Street address |
Largo R. Benzi, 10
|
| City |
Genova (GE) |
| ZIP/Postal code |
16132 |
| Country |
Italy |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE44099 |
NAC, Tiron and Trolox impair GBM tumorigenic cell survival by mechanisms independent from antioxidant activity |
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