|
| Status |
Public on Dec 01, 2008 |
| Title |
mouse colon_hnf4mutant_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
adult male mouse colon, Hnf4a mutant
|
| Organism |
Mus musculus |
| Characteristics |
strain:C57BL/6; VillinCRE-HNF4loxp; male; tissue colon; mutant
|
| Biomaterial provider |
Francois Boudreau
|
| Treatment protocol |
Injection of Ketamine/xylazine (300mg/kg; 40mg/kg) before sacrifice
|
| Growth protocol |
Mice were maintained in pathogen free environnement.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction with the ambion Totally RNA kit
|
| Label |
biotin
|
| Label protocol |
Target RNA is reverse transcribed into cDNA and in-vitro transcription is performed to generate biotin-labeled cRNA for subsequent hybridization.
|
| |
|
| Hybridization protocol |
The hybridization mixture was prepared by mixing 15 mg biotinylated probe with control oligonucleotide B2 (final concentration 50 pM; Affymetrix), herring sperm DNA (final concentration 0.1 mg/ml; Research Genetics), acetylated bovine serum albumin (final concentration 0.5 mg/ml; Gibco BRL Life Technologies) in a final volume of 300 ml of 13 MES hybridization buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20; all reagents from Sigma). The hybridization mixture was denatured for 10 minutes at 99 C, incubated for 5 minutes at 45 C, and spun for 5 minutes in a benchtop microcentrifuge. The microarray was warmed to room temperature and prehybridized in hybridization buffer for 10–20 minutes at 45 C. The prehybridization solution was removed and 150 ml of the hybridization mix was added to the array. The array and probe fragments were incubated at 45 C overnight (16–20 hours). After hybridization, nonspecifically bound probe was removed by washing with the GeneChip Fluidics station 400 (Affymetrix). In total, 10 low-stringency washes (63 SSPE, 0.01% Tween-20, 0.005% Antifoam) and 4 high-stringency washes (100 mM MES, 0.1 M NaCl, 0.01% Tween-20, 50 C) were performed (all reagents from Sigma).
|
| Scan protocol |
Specifically bound probe was detected by incubating the arrays with SAPE (Molecular Probes) and scanning the chips with a Hewlett-Packard GeneArray scanner (Affymetrix). A second scan was performed after signal enhancement with biotinylated anti-streptavidin antibody Vector Laboratories). The scanned images were analyzed using the GeneChip Analysis Suite 3.3 (Affymetrix) to identify genes differentially expressed among the RNA samples.
|
| Description |
Monitor the effect of HNF4alpha lost in mouse colonic epithelium
|
| Data processing |
RMA algorithm
|
| |
|
| Submission date |
Jun 11, 2008 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Francois Boudreau |
| E-mail(s) |
francois.boudreau@usherbrooke.ca
|
| Phone |
819-820-6876
|
| Fax |
819-564-5320
|
| Organization name |
University of Sherbrooke
|
| Department |
Anatomy and Cell Biology
|
| Lab |
Boudreau
|
| Street address |
3001 12e ave Nord
|
| City |
Sherbrooke |
| State/province |
Quebec |
| ZIP/Postal code |
J1H 5N4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE11759 |
Role of HNF4alpha in the adult colon |
|
| Relations |
| Reanalyzed by |
GSE119085 |
