|
| Status |
Public on May 18, 2016 |
| Title |
Liver_PALMdiet_rep02 |
| Sample type |
RNA |
| |
|
| Source name |
Liver, mouse, PALM diet, replicate 02
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
strain: C57BL/6J
age: 13 weeks
tissue: liver
diet: 5 weeks on High fat PALM diet
|
| Treatment protocol |
At 8 weeks of age, mice were randomly assigned to the experimental groups and fed during 5 weeks one of the two high fat diets containing palm oil (PALM diet; 55.6% SFA, 34.8% MUFA and 8.8% PUFA) or linseed oil (LIN diet; 9.7% SFA, 22.5% MUFA and 66.7% PUFA (49.7% ALA)). Diets were prepared freshly twice a week, stored at 4°C, and provided daily to mice as soft pellets. The calculated composition (in energy %) of both high fat diets is 16.7% protein, 32.1% carbohydrate, and 51.1% lipid.
|
| Growth protocol |
Seven-week old male C57BL/6J mice were purchased from Charles River (L’Arbresle, France). They were housed collectively on wood litter, at 23 ± 1°C under 12-h light/dark cycles (light on at 06:00 am) and were fed ad libitum a standard pelleted diet (SAFE A04, Augy, France) during acclimatization for 1 week.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Immediately following euthanasia, liver was removed and weighted. Liver samples of ~100 mg were snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA) following manufacturer's instructions. Total RNA quality was controlled on an Agilent 2100 Bioanalyzer (Agilent Technologies) and concentration was assayed using absorbance at 260nm on a Nanodrop ND-1000 spectrophotometer (Thermo Scientific)
|
| Label |
Cy3
|
| Label protocol |
For each sample, Cyanine-3 (Cy3) labeled cRNA was perpared from 1µg of total RNA using the One-color Quick Amp Labeling kit (Agilent Technologies) according to the manufacturer's instructions. Dye incorporation and cRNA yield were controlled on a Nanodrop ND-1000 spectrophotometer.
|
| |
|
| Hybridization protocol |
1,65 µg of Cy3-labelled cRNA was fragmented (60°C, 30 min) and hybridized to Agilent Mouse GE 4x44K v2 microarrays (G4846A) following the manufacturer's instructions. Following Hybridization, microarrays were washed sequentially in Wash buffer 1 (1 min), Wash buffer 2 (37°C, 1 min) and Stabilization and Drying Solution (30 sec) as recommended by the manufacturer.
|
| 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 026655_D_F_20100123). All subsequent data analyses were done under R (www.r-project.org) using Bioconductor packages (www.bioconductor.org). Raw data (median of pixels intensity for each spot) 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 14 out of 18 microarrays or with a weight of 1 in at least 8 microarrays from at least one experimental group. At this step, 18060 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 log normalized signal of each set of replicated probes. The resulting matrix has 18060 rows each corresponding to a unique ProbeName (provided as data Matrix).
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| |
|
| Submission date |
Sep 21, 2015 |
| Last update date |
May 20, 2016 |
| Contact name |
Pascal GP Martin |
| E-mail(s) |
pascal.martin@inrae.fr
|
| 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 |
GPL11202 |
| Series (1) |
| GSE73290 |
Hepatic transcriptome of mice fed high fat diets containing palm or linseed oil |
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