|
| Status |
Public on Jun 29, 2014 |
| Title |
PC-3 Gefitinib, biological rep1 [mRNA] |
| Sample type |
RNA |
| |
|
| Source name |
PC-3 cells, 60μM gefitinib (Gef), 24h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC-3
treatment: gefitinib
treatment duration: 24h
|
| Treatment protocol |
About 1.0x10^3 PC-3 cells plated in a petri dish (diameter, 3.5 cm) were incubated at 35°C overnight, then gefitinib (iressa), luteolin and/or resveratrol dissolved in DMSO were added into the medium.
|
| Growth protocol |
The PC-3 cells were provided by the Japanese Cancer Research Resources Bank (JCRB). The cells were maintained in 5% CO2 at 35°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone Laboratory), penicillin (100U/mL), and streptomycin (100μg/mL).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from PC-3 cells 24h following treatment with DMSO, 60μM luteolin (Lut), 60μM gefitinib (Gef) or their co-administration (Lut+Gef) using an miRNeasy Mini kit according to the manufacturer's instructions (Qiagen). The quality of the RNAs was estimated by using the RNA 6000 Nano LabChip Kit (p/n 5065-4476) on the Agilent 2100 Bioanalyzer (G2940BA; Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
100 ng of total RNAs were independently reverse-transcribed using oligo-dT primers containing the T7 RNA polymerase promoter sequence to generate cDNAs and AffinityScript, RTase, which were then subjected to in vitro transcription using T7 RNA polymerase to label the cRNAs with Cy3-CTP (Amersham Pharmacia Biotech, Piscataway, NJ) using a Low input Quick-Amp Labeling Kit (Agilent Technologies).
|
| |
|
| Hybridization protocol |
Before hybridization, 1650 ng labeled cRNA of each product was fragmented and mixed with control targets and hybridization buffer according to the manufacturer's protocol (Agilent Technologies). Hybridizations to Agilent-014850 arrays were done for approximately 17h at 65°C. The slides were washed according to the manufacturer's manual.
|
| Scan protocol |
Scanning of microarrays was performed with 5µm resolution using a DNA microarray laser scanner (Agilent Technologies). The array was scanned using Agilent G2505C DNA microarray scanner. Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).
|
| Description |
PC-3 Gefitinib 1
PC-3 cells treated for 24h with 60μM gefitinib (Gef).
|
| Data processing |
The Subio Platform and Subio Basic Plug-in (v1.15; Subio Inc., Aichi, Japan) was then used to calculate the between-sample fold change analyzed by one-sample t-test, Student T-test.
|
| |
|
| Submission date |
Dec 10, 2013 |
| Last update date |
Jun 29, 2014 |
| Contact name |
Daisuke Okuzaki |
| E-mail(s) |
dokuzaki@biken.osaka-u.ac.jp
|
| Phone |
+81-6-6879-4935
|
| Organization name |
Osaka univ.
|
| Department |
Immunology Frontier Research Center
|
| Lab |
Human Immunology （Single Cell Genomics)
|
| Street address |
Yamadaoka 3-1
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4133 |
| Series (2) |
| GSE53180 |
DNA microarray analysis following gefinitib/luteolin administration to PC-3 cells |
| GSE53181 |
Microarray analysis following gefinitib/luteolin administration to PC-3 cells |
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