|
| Status |
Public on Oct 01, 2012 |
| Title |
PFR31_1_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
NSCLC cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Non-Small Cell Lung Cancer (NSCLC)
pf299804: Resistant
|
| Treatment protocol |
N/A
|
| Growth protocol |
Cells were grown in RPMI supplemented with 10% FBS and antibiotics (PC9 and PFR3) or RPMI supplemented with 10% FBS, antibiotics and PF299804 (PFR3+). Cells were growing exponentially before the harvest
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using trizol and clened up with RNAeasy kit (Qiagen)
|
| Label |
biotin
|
| Label protocol |
At DFCI Microarray core, RNA was converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis was then carried out. The double-stranded cDNA was 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) was purified from the IVT reaction mixture using magnetic particles. The cRNA was quantified spectrophotometrically and purity of the cRNA was also assessed by spectrophometric measurements. Those cRNA's that fall outside of an acceptable range was not routinely carried forward in the analysis. The purified cRNA was fragmented in order to facilitate the subsequent hybridization step.
|
| |
|
| Hybridization protocol |
The fragmented cRNA ws added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C. The hybridization solution was then removed. The chips were transferred to a fluidics instrument that performs washes to remove cRNA that had not hybridized to its complementary oligonucleotide probe. The bound cRNA was then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors were then added using biotinylated anti-streptavidin antibody and additional SAPE.
|
| Scan protocol |
Each cRNA bound at its complementary oligonucleotide was excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions were captured. These measures provide the basis of subsequent biostatistical analysis. The data was transferred to a file-system from which it can be downloaded via a password-protected URL.
|
| Description |
PF299804 Resistant, maintained in normal media after the resistant cells are established
|
| Data processing |
Normalized and analyzed using GenePattern (http://www.broadinstitute.org/cancer/software/genepattern/), selecting RMA method
|
| |
|
| Submission date |
Jun 01, 2012 |
| Last update date |
Oct 01, 2012 |
| Contact name |
Pasi Janne |
| E-mail(s) |
pasi_janne@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Medical Oncology
|
| Street address |
44 Binney Street Dana 820
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE38404 |
Resistance to Irreversible EGFR Tyrosine Kinase Inhibitors through a Multistep Mechanism Involving the IGF1R Pathway |
|
