|
| Status |
Public on Oct 19, 2011 |
| Title |
NCI-H292-pcDNA_naive_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
NCI-H292 cells, pcDNA, 24hr, no treatment, replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: NCI-H292
overexpression: control
treatment: none
|
| Treatment protocol |
NCI-H292 cells, control and overexpressing HO-1, after reaching the confluence were treated with TNF (30 ng/ml) dissolved in RPMI 1640 medium deprived of serum. The cultures were incubated for 24 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Growth protocol |
NCI-H292 cells were cultured in RPMI-1640 medium suplemented with fetal bovine serum to a final concentration of 10%, at 37.0°C in 5% of CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the QIAzol Lysis Reagent (Qiagen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug cDNA (reversely transcribed from 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 |
Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x GEx Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven (10 rpm). 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 (G2505B US22502687) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel was set to Green and Green PMT. Two readings were performed: XDR Hi 100% and XDR Lo 10%).
|
| Description |
Gene expression after 24hr in non treated NCI-H292 control cells
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.1.1 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 014850_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
May 27, 2010 |
| Last update date |
Oct 19, 2011 |
| Contact name |
Slawomir Golda |
| Organization name |
Jagiellonian University
|
| Department |
Faculty of Biochemistry, Biophysics adn Biotechnology
|
| Street address |
Gronostajowa 7
|
| City |
Krakow |
| ZIP/Postal code |
30-387 |
| Country |
Poland |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE22030 |
HO-1 modulates the expression of genes involved in inflammation, angiogenesis, proliferation, apoptosis and cell adhesion in human lung cancer cells. |
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