|
| Status |
Public on Aug 31, 2011 |
| Title |
Tissue_model_Epi_200_0Gy_625_875_um_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
human foreskin, male, epidermis, keratinocytes
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Epi-200
tissue: epidermis model
treatment: 0Gy 625-875 um
|
| Biomaterial provider |
Mat-Tek Corporation, Ashland MA
|
| Treatment protocol |
Tissues were irradiated with 0 and 2.5Gy of protons
|
| Growth protocol |
Samples were supplied on ice with overnight delivery and were cultured 24 h prior irradiation and 16 h prior harvesting, postirradiation. Growth medium, NMM-100, was kindly supplied by the manufacturer. Medium was changed every 24h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with RNaqueous Kit (Applied Biosystems), tested for spectral characteristics (NanoDrop, Thermo Scientific) and electrophoretically (Bioanalyser 2100, Agilent).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Quick-Amp Labeling Kit (Agilent) according to the manufacturer’s instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent 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), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan setting with extended dynamic range, Scan area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10%).
|
| Description |
Source of irradiation: protons
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1.3.1 (Agilent) using default parameters (protocol GE1-v1_91_1 and Grid: 014850_D_20070207) to obtain background subtracted and spatially detrended green Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers or Features not Positive and significant were excluded
|
| |
|
| Submission date |
Aug 25, 2010 |
| Last update date |
Aug 31, 2011 |
| Contact name |
Alexandre Mezentsev |
| E-mail(s) |
am2710@columbia.edu
|
| Phone |
(212) 305-2166
|
| Fax |
(212) 305-7391
|
| Organization name |
Columbia University
|
| Department |
Dermatology
|
| Street address |
630 west 168th Street, VC 15-204
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10031 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE23803 |
Bystander response to 2.5 Gy of protons in a human 3-dimensional skin model in 16 h after exposure |
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