|
| Status |
Public on Jan 29, 2019 |
| Title |
p53+/+_dox_24h |
| Sample type |
RNA |
| |
|
| Source name |
HCT116 p53+/+, doxorubicin, 24 h.
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
genotype: p53+/+
treatment: doxorubicin
time to harvest: 24 hours
|
| Treatment protocol |
HCT116 p53+/+ and HCT116 p53−/− cells were treated with 2 μg/mL of doxorubicin for 2 hours. The cultures were incubated for 0, 12, 24, or 48 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the cells using RNeasy Plus Universal Mini Kits (Qiagen) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
The fluorescent cRNA using cyanine 3-labelled (Cy3) targets was generated following the Agilent’s One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp labeling) protocol. The Cy3-labeled cRNA was purified by Rneasy Mini Kit (Qiagen) and quantified by Nanodrop-2000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction containing Agilent Fragmentation Buffer and Gene Expression Blocking Agent following the manufacturers instructions. Hi-RPM Hybridization Buffer was added to stop the fragmentation. Samples were hybridized to SurePrint G3 Human Gene Expression 8x60K Microarrays for 17 hours at 65°C with rotation. After hybridization, microarrays were washed 1 minute at room temperature with Gene Expression Wash Buffer 1 and 1 minute with 37°C pre-warmed Gene Expression Wash Buffer 2.
|
| Scan protocol |
Slides were scanned on the Agilent Microarray Scanner using one color scan setting for 8x60K array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 24h in doxorubicin-treated wild-type p53 cells
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028004_D_F_20140813) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Jan 28, 2019 |
| Last update date |
Jan 29, 2019 |
| Contact name |
Koichi Matsuda |
| Organization name |
The University of Tokyo
|
| Street address |
4-6-1, Shirokanedai, Minato-ku
|
| City |
Tokyo |
| ZIP/Postal code |
108-8639 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE125787 |
Identification of p53-regulated genes in wild-type p53 or p53-null HCT116 cells |
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