|
| Status |
Public on Dec 14, 2011 |
| Title |
PB_Kidney_treated_11 |
| Sample type |
RNA |
| |
|
| Source name |
4 wk_Phenobarbital treatment_kidney
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6C3F1/Crl (C57BL/6 male x C3H/He female)
gender: male
tissue: Kidney
treatment: Phenobarbital
duration: 4 weeks
donor id: 11
|
| Treatment protocol |
Liver and kidney tissues were removed, split into several sections, frozen in liquid nitrogen and stored at −80°C.
|
| Growth protocol |
29–32 days old male B6C3F1/Crl (C57BL/6 ♂ x C3H/He ♀) mice were obtained from Charles River Laboratories (Germany). Animals were allowed to acclimatise for 5 days prior to being randomly divided into two treatment groups (n = 10) and phenobarbital (Sigma 04710, 0.05% (w/v) in drinking water) was administered to one group through ad libitum access to drinking water for 28 days. Mice were checked daily for activity and behavior and sacrificed on the last day of dosing (day 28).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen liver and kidney samples were homogenized in TRIzol reagent (Invitrogen) and subsequently purified on a silica-gel-based-membrane (RNeasy, Qiagen) according to the manufacturer's instructions. RNA quality was assessed by measuring the RIN (RNA Integrity Number) using an Agilent 2100 Bioanalyzer. RNA was stored at −80°C until GeneChip® experiment analysis.
|
| Label |
biotin
|
| Label protocol |
Processing of GeneChip® experiments was conducted as recommended by the manufacturer of the GeneChip® system (Affymetrix). For tissue samples, double stranded cDNA was synthesized with a starting amount of 0.1 µg total RNA. For RNA reverse transcription, the GeneChip® 3′ IVT Express Labelling Assay (lot ID 0904012, Affymetrix) was used in the presence of a T7-(dT)24 DNA oligonucleotide primer (Affymetrix). The cDNA was then transcribed in vitro in the presence of biotinylated ribonucleotides to form biotin-labelled amplified RNA (aRNA). The labelled aRNA was then purified and quantified by UV spectrophotometry at 260 nm and fragmented.
|
| |
|
| Hybridization protocol |
The arrays were washed and stained with the GeneChip® Hybridization Wash and Stain kit (Affymetrix). The washing and staining steps were performed with GeneChip® Fluidics Workstation 450 (Affymetrix).
|
| Scan protocol |
Arrays were scanned using a solid-state laser scanner (GeneArray® Scanner 3000 combined with the GeneChip® autoloader, Affymetrix).
|
| Description |
706087
|
| Data processing |
The Affymetrix GeneChip® Operating Software (GCOS) was used to generate the primary and secondary raw data files. Affymetrix chips were normalized with the MAS5 method (scaled to a trimmed mean of 150 per chip, Affymetrix, I. (2002) Statistical Algorithms Description Document http://www.affymetrix.com/support/technical/whitepapers.affx). For gene expression analysis, we excluded all probe sets with an average MAS5 signal of less than 50 in both groups (liver control and treated) to avoid large fold changes in genes that are not expressed under both of the experimental conditions. The cut-off of 50 is empirical, and we found that it performs better than the suggested present/absent calls of MAS5. This reduces the number of probe sets from 45,037 to 18,981 probes.
|
| |
|
| Submission date |
Dec 13, 2011 |
| Last update date |
Jun 08, 2015 |
| Contact name |
Jonathan Moggs |
| E-mail(s) |
[email protected]
|
| Organization name |
Novartis
|
| Street address |
Fabrikstrasse 2
|
| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL1261 |
| Series (3) |
| GSE34423 |
Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice [Expression array]. |
| GSE34463 |
Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice. |
| GSE68387 |
IMI MARCAR Project: towards novel biomarkers for cancer risk assessment |
|
