|
| Status |
Public on Nov 07, 2012 |
| Title |
Liver_WildType_NormalDiet_2 |
| Sample type |
RNA |
| |
|
| Source name |
Liver, wild-type control, normal diet
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: mixed Sv129/C57Bl6J
genotype: wild-type
gender: male
age: 28 weeks
tissue: liver
treatment: normal diet
|
| Treatment protocol |
Groups of mice were fed ad libitum for 7 weeks a normal chow (2016 Teklad Global 16% Protein Rodent Diet, Harlan, France) or a Western Diet (TD.88051 Cocoa Butter Diet and Purina Mouse Chow, Harlan, France).
|
| Growth protocol |
28-week-old wild-type and LXR-/- mice were housed at 22+2°C on a 12 hour light/dark cycle and allowed free access to water and food. The in vivo study was conducted under the European Union Guidelines for the use and care of laboratory animals and were approved by an independent ethics committee.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Immediately following euthanasia, the liver was removed, weighed, dissected (~100 mg fragments from the large lobe), snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction. Total RNA was extracted from frozen liver samples with TRIzol reagent (Invitrogen) following the manufacturer's instructions. Total RNA samples were controlled for integrity on an Agilent 2100 Bioanalyzer (Agilent Technologies) and assayed at 260nm on a Nanodrop ND-1000 spectrophotometer (Thermo Scientific).
|
| Label |
Cy3
|
| Label protocol |
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 1 µg of total RNA using the One-Color Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Courtaboeuf, France). Dye incorporation and cRNA yield were checked with a NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 µg of Cy3-labelled cRNA (specific activity >12.8 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min), Wash buffer 2 (Agilent Technologies, 37°C, 1 min) and Stabilization and Drying Solution (Agilent Technologies, 30 sec).
|
| Scan protocol |
Slides were scanned immediately after washing on a GenePix™ 4000B array scanner.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_95 and Grid: 014868_D_20070207). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot):
myfunw<-function(x)
{okType<-x$ControlType==0
okFoundGreen<-x$gIsFound==1
okPos=x$gIsPosAndSignif==1
okWellAbove<- x$gIsWellAboveBG==1
as.numeric(okType & okFoundGreen & okPos & okWellAbove)}
We selected the spots with a weight of 1 for 16 out of 20 microarrays or with a weight of 1 in all 5 microarrays from at least one experimental group. At this step, 26057 spots out of 45018 were selected. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 24493 rows, each corresponding to a unique ProbeName.
|
| |
|
| Submission date |
May 21, 2012 |
| Last update date |
Nov 07, 2012 |
| Contact name |
Pascal GP Martin |
| E-mail(s) |
[email protected]
|
| Organization name |
INRAE
|
| Department |
UMR1332 BFP
|
| Lab |
FDFE
|
| Street address |
71 avenue Edouard Bourlaux
|
| City |
Villenave d'Ornon |
| ZIP/Postal code |
33140 |
| Country |
France |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE38083 |
Hepatic transcriptome in wild-type and LXR-deficient mice |
|
