|
| Status |
Public on Oct 16, 2009 |
| Title |
0,05_2h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
A549 cell line isolated from a human non small cell lung cancer, treated in vitro with various concentrations of triptolide
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
gender: male
age: 58
tissue: lung cancer
|
| Biomaterial provider |
ATCC, American Type Culture Collection: ATCC Data sheet for the A549 cell line: http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CCL-185&Template=cellBiology
|
| Treatment protocol |
A549 cells were seeded in 75cm2 plates up to 70% confluence before exposure to various concentrations of triptolide for 1, 2 and 4 hours before RNA was extracted.
|
| Growth protocol |
A549 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (65 µg/ml) and streptomycin (100 µg/ml).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed using TRIzol reagent (Invotrogen). Total RNA were dissolved in 20 µl of Rnase-free water and qualified using the NanoDrop ND-1000 Spectrophotometer
|
| Label |
Cy3
|
| Label protocol |
Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies) generates fluorescent cRNA with 500ng of total RNA. In this procedure, samples are labelled with cyanine 3. A primer containing poly dT and a T7 polymerase promoter is annealed to the poly A+ RNA. Reverse transcriptase is added to the reaction to synthesize the first and second strands of cDNA. Next, cRNA are synthesized from the double-stranded cDNA using T7 RNA polymerase, which simultaneously incorporates cyanine-labelled CTP.
|
| |
|
| Hybridization protocol |
1650 ng of cyanine 3-labelled amplified RNA are hybridised at 65°C for 17 hours
|
| Scan protocol |
Scanning is performed with the Agilent scanner using default parameters for 4x44 K formats. For an XDR extraction, two image files (.tif) are generated, one noted H (high, PMT 100) and the other one noted L (low, PMT 10)
|
| Description |
Sample corresponding to a treatment of 0,05µM of triptolide. Measurement after 2h. Replicate 2.
|
| Data processing |
Data are extracted with the Feature Extraction 9,1 software (Agilent Technologies). This software reads and processes raw microarray image files to prepare them for analysis. Feature extraction automatically assigns a grid template and a protocol, based on the barcode of the slide. The software finds microarray grids, determines feature intensities, rejects outliers, and calculates statistical confidences
|
| |
|
| Submission date |
Jun 23, 2009 |
| Last update date |
Oct 16, 2009 |
| Contact name |
Stéphane VISPE |
| E-mail(s) |
stephane.vispe@pierre-fabre.com
|
| Organization name |
Laboratoires Pierre Fabre
|
| Street address |
3 Rue des Satellites
|
| City |
Toulouse |
| ZIP/Postal code |
31000 |
| Country |
France |
| |
|
| Platform ID |
GPL8755 |
| Series (1) |
| GSE16760 |
Whole genome analysis in A549 cells treated for short incubation periods with various concentrations of triptolide |
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