|
| Status |
Public on Jul 01, 2011 |
| Title |
4h10R_3 |
| Sample type |
RNA |
| |
|
| Source name |
EPI200 4h 10 cGy
|
| Organism |
Homo sapiens |
| Characteristics |
cell model: Mat-Tek EPI-200 3-dimensional skin model
|
| Treatment protocol |
For irradiation, samples were installed on a carousel and exposed to 0, 0.1 or 2.5 Gy 4.5 MeV protons using the track segment irradiation facility of the 5.5-MV Singletron accelerator at the Radiological Research Accelerator Facility (RARAF) of Columbia University. After irradiation, samples were returned to plates filled with fresh culture medium and cultured for 4, 16 or 24h.
|
| Growth protocol |
EPI-200 was purchased from MatTek (Ashland, MA). At arrival, tissue plates were allowed to equilibrate to room temperature for 1h and then tissues were transferred to 6-well tissue culture dishes filled with NMM-100 medium (0.9ml/well) and cultured according to the manufacturer's instructions as previously described (Curren et al., 2006. PMID: 16781186).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the RNAqueous Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions and stored at -80°C. Briefly, the procedure included homogenization in a Potter homogenizer, binding to a spin column and on-column treatment with DNase I. The impurities were washed out and RNA was eluted with a commercial elution buffer preheated to 75°C. RNA yield and quality were assessed with the NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA) and the Bioanalyzer 2100 (Agilent Technologies, Foster City, CA).
|
| Label |
Cy3
|
| Label protocol |
RNA (100 ng of each sample) was labeled and amplified with the one-color Quick Amp Labeling Kit. Briefly, the procedure included reverse transcription and synthesis of cRNA covalently labeled with Cy3. The cRNA was purified with RNeasy (Qiagen, Valencia, CA) following the Agilent protocol and checked to ensure incorporation of > 8.0 pmol of Cy3 per μg of cRNA prior to fragmentation and hybridization
|
| |
|
| Hybridization protocol |
Hybridization to Agilent Whole Human Genome Oligo Microarrays (G4112A) was carried out in an Agilent hybridization oven for 17 h at 65°C in Agilent GE buffer according to the manufacturer's protocol.
|
| Scan protocol |
Hybridized slides were washed following Agilent's recommendations and then dried by centrifugation (1,500 rpm; 10 min) and immediately scanned with the Agilent DNA Microarray Scanner (G2505B) at 5 μm resolution and 100% PMT.
|
| Description |
EPI200 4h 10 cGy 3
|
| Data processing |
Scanned images were analyzed with default parameters using Feature Extraction Software 9.1 for background correction and flagging of non-uniform features. Background corrected hybridization intensities were imported into BRB-ArrayTools, Version 3.7.1 (Simon et al., 2007. PMID:19455231) log2-transformed and median normalized.
|
| |
|
| Submission date |
Jul 02, 2009 |
| Last update date |
Jul 01, 2011 |
| Contact name |
Sally Amundson |
| E-mail(s) |
saa2108@cumc.columbia.edu
|
| Organization name |
Columbia University
|
| Department |
Center for Radiological Research
|
| Street address |
630 W. 168th St
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE16935 |
Response of the EPI-200 human 3-D skin model to high and low doses of protons |
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