|
| Status |
Public on Dec 22, 2005 |
| Title |
8rightB |
| Sample type |
RNA |
| |
|
| Source name |
ear tissue
|
| Organism |
Mus musculus |
| Characteristics |
CD1 male mice 25-35 g
|
| Biomaterial provider |
Charles River Laboratories
|
| Treatment protocol |
6.25 mg Dimercaprol 15 min before 0.08 mg Sulfur Mustard
24 hr
|
| Growth protocol |
A single 5 microL application of SM (0.08 mg dose, 0.5 micromoles) in methylene chloride was applied topically to the inner surface of the right ear. The left ear (vehicle control) was exposed to 5 microL of methylene chloride only. For drug treatment, animals were administered 10 microL of dimercaprol (6.25 mg dose, 50.3 micromoles), 30 microL of OHV (0.585 mg dose, 1.995 micromoles), or 20 microL of indomethacin (1.34 mg dose, 3.74 micromoles) in ethanol 15 minutes prior to SM challenge. A group of animals received only drug on the right ear and ethanol (drug vehicle) on the left ear. A group of untreated, unexposed animals served as naïve controls. At 24 hr post-exposure, animals were euthanized and an 8 mm diameter skin biopsy was obtained. The skin biopsy was weighed and immediately frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from frozen mouse ear biopsies using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The quality and amount of RNA was monitored throughout processing with an Agilent Bioanalyzer (Agilent, Palo Alto, CA) and a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Nanodrop Technologies, Rockland, DE).
|
| Label |
biotin and streptavidin/phycoerythrin
|
| Label protocol |
10 µg of total RNA was used to generate first-strand cDNA by using a T7-linked oligo(dT) primer. After second-strand synthesis, in vitro transcription was performed with biotinylated nucleotides (Enzo Kits from Affymetrix), resulting in approximately 100-fold amplification of cRNA. The target cRNA generated from each sample was processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (http://www.affymetrix.com/support/technical/ manual/expression_manual.affx).
|
| |
|
| Hybridization protocol |
Spiked controls were added to 15 µg of fragmented cRNA before overnight hybridization using 10 µg of cRNA.
|
| Scan protocol |
Arrays were washed and stained with streptavidin-phycoerythrin before being scanned on an Agilent GeneArray Scanner driven by Microarray Suite 5.0 software (Affymetrix). After scanning, array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches on the chip surface.
|
| Description |
sulfur mustard exposed mouse ear
|
| Data processing |
Arrays were then washed and stained with streptavidin-phycoerythrin before being scanned on an Agilent GeneArray Scanner. After scanning, array images were assessed by eye to confirm scanner alignment and the absence of significant bubbles or scratches on the chip surface. All arrays were scaled to a target intensity of 150 (using Affymetrix Microarray Suite 5.0 array analysis software), and internal controls were assessed to determine array quality.
|
| |
|
| Submission date |
Jul 15, 2005 |
| Last update date |
Dec 22, 2005 |
| Contact name |
James F Dillman |
| E-mail(s) |
james.f.dillman2.civ@mail.mil
|
| Phone |
4104361723
|
| Organization name |
U.S. Army Medical Research Institute of Chemical Defense
|
| Department |
Cell and Molecular Biology Branch
|
| Lab |
Molecular Toxicology Team
|
| Street address |
3100 Ricketts Point Rd
|
| City |
Aberdeen Proving Ground |
| State/province |
MD |
| ZIP/Postal code |
21010-5400 |
| Country |
USA |
| |
|
| Platform ID |
GPL340 |
| Series (1) |
| GSE2950 |
Microarray analysis of mouse ear tissue exposed to bis-(2-chloroethyl) sulfide |
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