|
| Status |
Public on Jun 29, 2014 |
| Title |
PC-3 DMSO, biological rep2 [miRNA] |
| Sample type |
RNA |
| |
|
| Source name |
PC-3 cells, DMSO, 24h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC-3
treatment: DMSO
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 aliquots of total RNA was used for making miRNA probes according to the Agilent protocol (ver. 2.4). Briefly, total RNA was dephosphorylated with calf intestine alkaline phosphatase, denatured with dimethyl sulfoxide and labeled with Cyanine 3-pCp using T4 RNA ligase using the miRNA Labeling Kit.
|
| |
|
| Hybridization protocol |
Probes were hybridized to Agilent-041686 arrays using miRNA Hybridization Kit at 55°C for 20 hours with rotation. Then, the slides were washed by Gene Expression Wash Buffer 1 at room temperature for 5 minutes and by Gene Expression Wash Buffer 2 at 37°C for 5 minutes.
|
| Scan protocol |
After hybridization and washing, the slides were scanned using an Agilent scanner (G2505C). Images were extracted using Agilent Feature Extraction software (ver. 10.7.3).
|
| Description |
PC-3 DMSO_2
PC-3 cells treated for 24h with DMSO.
|
| Data processing |
Data were processed by the Agilent GeneSpring GX software (ver. 12.6). Total gene signal from GeneView data files extracted with default settings in Agilent Feature Extraction was used as signal for miRNAs. Differences in miRNA expressions were normalized using the PC-3 treated DMSO sample as a control and determined as if the fold change of expression values was >2.0 and the p value was <0.05 using Student's t test for further analysis.
|
| |
|
| 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 |
GPL18046 |
| Series (2) |
| GSE53178 |
miRNA array analysis following gefinitib/luteolin administration to PC-3 cells |
| GSE53181 |
Microarray analysis following gefinitib/luteolin administration to PC-3 cells |
|
