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| Status |
Public on Mar 04, 2014 |
| Title |
lean_1mg/L_mouse#5 |
| Sample type |
RNA |
| |
|
| Source name |
liver tissue from treated lean animal (1mg/L), mouse/replicate #5
|
| Organism |
Mus musculus |
| Characteristics |
background strain: C57BL/6J
tissue: liver
gender: male
genotype: wild type
dose: 1 mg/L
treatment duration: 4 months
|
| Treatment protocol |
After 1 week of acclimatization, mice were further split into 4 subgroups (n=6 mice per group) that were treated or not with a mixture of 11 drugs at a concentration of 1 mg/L (for each drug). All these drugs (acetaminophen, caffeine, carbamazepine, cotinine, diclofenac, erythromycin, ibuprofen, phenazone, roxithromycin, salicylic acid and sulfamethoxazole) were purchased from Sigma-Aldrich (St. Quentin-Fallavier, France). Considering the molecular weight of the molecules, 1 mg/L corresponded to concentrations ranging from 1.2 µmol/L for roxithromycin to 7.2 µmol/L for salicylic acid. Mice were exposed during 4 months to these drugs by way of the drinking water, which was renewed every week.
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| Growth protocol |
Five-week-old male C57BL/6J-+/+ mice (wild-type, also referred to as lean mice) weighing 19 to 20 g and C57BL/6J-ob/ob mice, weighing 28 to 32 g, and were purchased from Janvier (Le-Genest-St-Isle, France) and housed in the animal house facility of Rennes 1 University under a 12 h light-dark cycle. All mice were fed ad libitum on a normal diet bringing 2820 kcal per kg of food (A04 biscuits; UAR, Villemoisson-sur-Orge, France).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were then sacrificed by cervical dislocation and liver was quickly removed. While a majority of the liver fragments were immediately frozen in liquid nitrogen some of them were rapidly processed for appropriate histological staining. Collected tissues frozen in liquid nitrogen were subsequently stored at -80°C until total RNA extraction. All experiments were performed according to national guidelines for the use of animals in biomedical research and approved by the local Ethics Committee in Animal Experiment of Rennes 1 University and Robert Debré Hospital. Total RNA was extracted from liver samples using a standard RNA extraction protocol (Trizol) and quality-checked with an Agilent 2100 Bioanalyzer platform. All RNA samples revealed RNA Integrity Number (RIN) values between 7.2 and 8.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was amplified and labeled with Cy3 fluorescent dye using Agilent one color low-input QuickAmp labeling kit following according to the manufacturer's instructions. Starting material was 1µg of total RNA. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 1.65 μg Cy3-labeled fragmented cRNA was hybridized overnight (17 hours, 65 °C) to Agilent Whole Mouse Genome Oligo Microarrays 4x44K using Agilent’s recommended hybridization chamber and oven. Microarrays were then washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min. The last washing step was performed with acetonitrile. Finally, fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies).
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| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray C Scanner (G2505C) using one color scan setting for 4x44k array slides (scan resolution 5µm).
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| Description |
Gene expression profiling of liver tissue from WT mouse treated with 1 mg/L drug mixture during 4 months
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| Data processing |
The scanned images were analyzed with Feature Extraction Software version 10.5.1.1 (Agilent Technologies) using default parameters (protocol GE1_105_Jan09 and Grid: 014868_D_F_20070820). Gene expression data were further analyzed by using the GeneSpring software (Agilent Technologies). Normalization was performed with the 75th percentile shift algorithm. A baseline transformation to the median of all samples was applied. Filtration by flag and signal intensity was applied. Briefly, were retained only the entities in which at least 50% of the values had a detected flag (i.e. a positive and significant feature as defined by GeneSpring). For the filtration by signal intensity, were retained the entities in which at least 50% of the values were within the range of interest (i.e. 40-100th percentile).
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| |
|
| Submission date |
Jul 25, 2013 |
| Last update date |
Mar 04, 2014 |
| Contact name |
Cedric Coulouarn |
| E-mail(s) |
[email protected]
|
| Organization name |
INSERM
|
| Department |
U1242
|
| Lab |
COSS
|
| Street address |
CLCC Eugène Marquis, Rue de la Bataille Flandres Dunkerque, Bat D, 1er étage
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE49195 |
Gene expression profiling of liver tissue from lean (WT) and obese (ob/ob) mice following a chronic exposure of low doses of pharmaceuticals |
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