|
| Status |
Public on Apr 19, 2007 |
| Title |
Carboplatin treated cells Rep2 |
| Sample type |
RNA |
| |
|
| Source name |
NSCLC adenocarcinoma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
A549 NSCLC cell line
|
| Biomaterial provider |
ATCC/Dr. Barrett Rollins
|
| Treatment protocol |
Cells were cultured overnight in DMEM 10% FBS (pen/strep). Media was changed with cells receiving either control media, 1 micromolar rosiglitazone, 10 micromolar carboplatin or combination of carboplatin and rosiglitazone.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were scraped into trizol and transfered to phaselock tubes. RNA was extracted according to manufactures directions.
|
| Label |
biotinylated
|
| Label protocol |
RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out.
The double-stranded cDNA is used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates.
The biotinylated complementary RNA (cRNA) is purified from the IVT reaction mixture using the RNeasy system (Qiagen).
|
| |
|
| Hybridization protocol |
The purified cRNA is fragmented in order to facilitate the subsequent hybridization step. The cRNA is purified from the fragmentation reaction using phenol/chloroform extraction and ethanol precipitation.
The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C.
The chips are then transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
|
| Scan protocol |
Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured.
|
| Description |
Nothing more to add
|
| Data processing |
Array normalization, expression value calculation and clustering analysis were performed using DNA-Chip Analyzer. The Invariant Set Normalization method was used to normalize arrays at probe cell level to make them comparable, and the model-based method was used for probe-selection and computing expression values.
|
| |
|
| Submission date |
Feb 14, 2007 |
| Last update date |
Feb 20, 2007 |
| Contact name |
Geoffrey Girnun |
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Cancer Biology
|
| Street address |
One Jimmy Fund Way
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE7035 |
Synergy between PPARgamma ligands and platinum-based drugs in cancer |
|
