|
| Status |
Public on Apr 14, 2015 |
| Title |
PC9 gefitinib resistant_2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
PC9 cell line resistant to gefitinib
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC9
population: resistant to gefitinib
treatment: Gefitinib (1.5 uM), dose escalation
|
| Treatment protocol |
Cells were harvested when they had grown to 80% confluency. The cells were washed once in 10mls of PBS and then pelleted at 1000rpm for 5 minutes. The supernatant was removed and the cell pellet frozen at -80oC until DNA preparation was performed.
|
| Growth protocol |
Resistant cell populations were cultured in RPMI, 10% FCS, 2mM L-glutamine supplemented with the indicated EGFR inhibitor. Parental cells were cultured in RPMI, 10% FCS, 2mM L-glutamine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was prepared from the cell pellets using the Allprep DNA/RNA/miRNA Universal kit (Qiagen, Valencia, CA)
|
| Label |
Cy3
|
| Label protocol |
Genomic DNA extracted from experimental (cell lines) and normal control female gDNA (Promega, Madison, WI) samples was labeled using CytoSure HT Genomic DNA Labelling Kit (Oxford Gene Technology, Oxfordshire, UK) following the OGT protocol for aCGH analysis with an adjustment to the post purification step. After labelled targets were combined in one tube, an additional 2 μl of water was added and directly proceeded to hybridization.
|
| |
|
| Channel 2 |
| Source name |
normal female gDNA, Promega
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
phenotype: normal
|
| Treatment protocol |
Cells were harvested when they had grown to 80% confluency. The cells were washed once in 10mls of PBS and then pelleted at 1000rpm for 5 minutes. The supernatant was removed and the cell pellet frozen at -80oC until DNA preparation was performed.
|
| Growth protocol |
Resistant cell populations were cultured in RPMI, 10% FCS, 2mM L-glutamine supplemented with the indicated EGFR inhibitor. Parental cells were cultured in RPMI, 10% FCS, 2mM L-glutamine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was prepared from the cell pellets using the Allprep DNA/RNA/miRNA Universal kit (Qiagen, Valencia, CA)
|
| Label |
Cy5
|
| Label protocol |
Genomic DNA extracted from experimental (cell lines) and normal control female gDNA (Promega, Madison, WI) samples was labeled using CytoSure HT Genomic DNA Labelling Kit (Oxford Gene Technology, Oxfordshire, UK) following the OGT protocol for aCGH analysis with an adjustment to the post purification step. After labelled targets were combined in one tube, an additional 2 μl of water was added and directly proceeded to hybridization.
|
| |
|
| |
| Hybridization protocol |
The hybridization master mix was prepared by mixing 25 μl of Cot-1 (1mg/ml), 26 μl or Agilent 1-x Blocking Agent and 130 μl of Aligent 2x HiRPM Hybridization Buffer. The master mix was mixed with labeled experimental and control DNA and hybridized to the Agilent Human Genome CGH 2x400k Miscoarray (Agilent Technologies, Santa Clara, CA). The arrays were incubated for 40 hours at 65°C in a rotating oven at 20 rpm. After hybridization slides were washed as stated in the protocol.
|
| Scan protocol |
All hybridized arrays were scanned on an Agilent G2505B scanner.
|
| Description |
Populations of PC9 cells resistant to gefitinib (1.5 uM)
|
| Data processing |
Images were analized using Agilent Feature Extraction Software (version 10.7.3.1). Nexus Copy Number software v7.0 was used for data processing and visualization. Log2 ratio for each gene was calculated as mean of log2 values of all probes mapped to a particular gene.
|
| |
|
| Submission date |
Jul 09, 2014 |
| Last update date |
Apr 15, 2015 |
| Contact name |
Aleksandra Markovets |
| Organization name |
AstraZeneca
|
| Department |
Oncology, iMED
|
| Lab |
Bioinformatics
|
| Street address |
35 Gatehouse Dr.
|
| City |
Waltham |
| State/province |
Massachusetts |
| ZIP/Postal code |
02451 |
| Country |
USA |
| |
|
| Platform ID |
GPL9777 |
| Series (1) |
| GSE59239 |
Acquired resistance to mutant-selective EGFR inhibitor AZD9291 is associated with increased dependence on RAS signaling |
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