|
| Status |
Public on Jun 11, 2007 |
| Title |
Cnr1-/-/Cnr2-/- female number 1, DNFB-treated allergic ear (#10_Ko-Tr-4) |
| Sample type |
RNA |
| |
|
| Source name |
whole ear tissue, DNFB-treated allergic ear 48h after 2nd challenge
|
| Organism |
Mus musculus |
| Characteristics |
Cnr1-/-/Cnr2-/- female number 1, 2 months old, whole ear tissue, DNFB-treated allergic ear
|
| Treatment protocol |
DNFB (1-fluoro-2,4 dinitrobenzene, Merck) was diluted in acetone/olive oil (4:1) immediately before use. Mice without any skin lesions and ear tags were sensitized by painting 50 µl of 0.2% DNFB on the shaved abdomen on two consecutive days. Controls were treated with 50 µl acetone-olive oil. For elicitation of CHS, ears of mice were painted with 10 µl of 0.3% DNFB on day 5. Ear thickness was measured 24 h, 48 h and 72 h after challenge using an engineers micrometer (Oditest, Fa. Kroeplin, Schlüchtern, Germany) and ear swelling was calculated in each mouse as the difference in ear thickness between the unchallenged control (left) and the challenged (right) ear. Experimental mice of either wildtype C57BL/6J line or CB1/CB2 double knockout mice (Cnr1-/-/Cnr2-/-) were used in the age of 8-10 weeks. Right ears of the mice were treated with DNFB, whereas left ears were untreated and used as control. All experimental animals were females. DNFB treated and control ears from three Cnr1-/-/Cnr2-/- and three Cnr1+/+/Cnr2+/+ mice were used for the gene expression analysis. Whole ear tissue was harvested 48h after the 2nd DNFB-challenge and immediately snap frozen in liquid nitrogen. Tissue was stored on –80°C until RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol (Invitrogen) and purified using RNeasy columns (Qiagen). RNA intergrity was assessed using Agilent 2100 Bioanalyzer (www.agilent.com). Five μg of total RNA was converted into double-stranded cDNA using T7-oligodT coupled primers. Ten μg of fragmented cRNA samples were hybridized to MG_430 2.0 Affymetrix GeneChips following the manufacturer´s instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2004, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on MG_430 2.0 Affymetrix GeneChips . GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
We used an Affymetrix GeneChip® Scanner 3000.
|
| Description |
ko mouse number 1
|
| Data processing |
Microarry expression values were generated using GC-RMA using software ArrayAssist® 3.0 (Stratagene). The GC-RMA-obtained log2-transformed expression values were imported into searchable database using this software.. For the treatment effects, which were independent of the genotype, all control (n=6) and DNFB-treated ears (n=6) were statistically analyzed using t-test with p ≤ 0.05 after Bonferroni correction and a differential expression higher than fourfold (given as an average log 2 ratio; ALR). The genotype effects were obtained by comparison of DNFB-treated ears of Cnr1+/+/Cnr2+/+ (n=3) versus Cnr1-/-/Cnr2-/- (n=3) with p ≤ 0.001 and ALR ≥ 1. Results for treatment and genotype effects were compared using Venn diagram.
Hierarchical clustering was performed on log2 transformed values using “ArrayAssist 3.0” (Stratagene) in two dimensions (e.g. both genes and samples).
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| |
|
| Submission date |
May 03, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Meliha Karsak |
| Organization name |
University of Bonn
|
| Department |
Molecular Psychiatry
|
| Street address |
Sigmund-Freud-Str. 25
|
| City |
Bonn |
| ZIP/Postal code |
53127 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE7694 |
Cannabinoid receptor double knockout mice (Cnr1 -/- /Cnr2 -/-) in CHS model |
|
| Relations |
| Reanalyzed by |
GSE119085 |
