|
| Status |
Public on Sep 21, 2012 |
| Title |
HeLa_hypoxia_IL_siHexim |
| Sample type |
RNA |
| |
|
| Source name |
HeLa, hypoxia and IL-1 for 1 hr, HEXIM1 siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
|
| Treatment protocol |
HeLa cells were transfected with double-stranded siRNA oligonucleotides directed against HEXIM1 (Thermo Scientific Dharmacon; siHex) or control siRNA (Santa Cruz; siScr) and 48 hrs later treated for 1 hr with IL-1beta in normoxic (N IL-1) or hypoxic (H IL-1) (5% CO2, 0.5% O2, balanced with N2) environment.
|
| Growth protocol |
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRI-zol reagent (Invitrogen). Pooled RNA from two replicates was used per array. Total RNA was isolated using RNeasy mini kit (Qiagen). Double-stranded cDNA was synthesised from 100ng of totalRNA using poly dT primer.
|
| Label |
Cy3
|
| Label protocol |
cRNA was synthesized using T7 RNA polymerase and labeled with Cyanine 3 dye (Low input Quick Amp labeling kit, one-color, Agilent Technologies) followed by purification with Rneasy mini kit (Qiagen).
|
| |
|
| Hybridization protocol |
Sample fragmentation was performed at 60°C for 30 minutes.On completion of the fragmentation reaction, samples were hybridized to Agilent SurePrint G3 Human GE microarrays (G4851A) at 65°C, 10rpm for 17 hours in a rotating 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 (G2539A) using Scan Control, version A.8.5.1, Agilent Technologies software. Scanning parameters were as follow: Dye channel - green, Scan region - Agilent HD (61x21.6 mm), TIFF file dinamic range - 20bit, R/G PMT gain - 100%, Scan resolution 3um.
|
| Description |
Gene expression in cells transfected with Hexim1 siRNA and treated with IL-1beta and hypoxia for 1 hr.
|
| Data processing |
The log2 transformed intensities were normalized on 75th percentile in each sample, and then turned into log-ratios against the control sample (N siScr).
|
| |
|
| Submission date |
Sep 20, 2012 |
| Last update date |
Aug 05, 2013 |
| Contact name |
Olga Sergeevna Safronova |
| E-mail(s) |
olga.cell@tmd.ac.jp
|
| Phone |
81-3-5803-5577
|
| Fax |
81-3-5803-0212
|
| Organization name |
Tokyo Medical and Dental University
|
| Department |
Cellular Physiological Chemistry
|
| Street address |
Bunkyo-ku, Yushima, 1-5-45
|
| City |
Tokyo |
| ZIP/Postal code |
113-8549 |
| Country |
Japan |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE41023 |
Transcriptional profiling of hypoxic cells and identification of HEXIM1-dependent genes. |
|
