|
| Status |
Public on Jul 01, 2012 |
| Title |
PC9 GR1 DMSO |
| Sample type |
RNA |
| |
|
| Source name |
PC9 GR1
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC-9
gefitinib-resistant: yes
treatment: DMSO
|
| Treatment protocol |
For RNA collection, 8 × 10^4 cells were seeded per well in 6-well plates and allowed to attach for 24 hours. The cells were then treated with 1uM gefitinib, 48hrs later we collected total RNA from these cell line.
|
| Growth protocol |
PC9 cell lines were cultured by RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cultures were incubated at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNAeasy kit (QIAGEN, , Valencia, CA) 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 175 ng RNA using Low Input Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) 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.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ml containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 8x60K human array (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting.
|
| Description |
Gene expression after 48hrs in 0uM Gefitinib human lung cancer cell line
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters (protocol GE1_1010_Sep10) 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 29, 2012 |
| Last update date |
Jul 01, 2012 |
| Contact name |
Junko Hamamoto |
| E-mail(s) |
junko_hamamoto@a7.keio.jp
|
| Organization name |
School of Medicine, Keio University
|
| Department |
Pulmonary Medicine
|
| Street address |
35 Shinanomachi, Shinjuku-ku
|
| City |
Tokyo |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE38302 |
Analysis of gene expression change by gefitinib treatment in gefitinib sensitive PC9 and resistant PC9. |
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