|
| Status |
Public on Oct 31, 2022 |
| Title |
Liver - Male_PPARaLKO_Pemafibrate - Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57B6/J
tissue: liver
gender: Male
genotype: LKO
treatment: Pemafibrate
washbatch: 1
|
| Treatment protocol |
Mice either received the PPARα agonist pemafibrate (0,1 mg/kg/day) or vehicle (Carboxymethyl cellulose 0,5%) by gavage for 14 days.
|
| Growth protocol |
Mice were housed at 20-22°C under 12h/12h light/dark conditions and fed a chow diet.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs were extracted from liver samples in males and females mice using the method Chomczynski P and Sacchi N (1987) with Tri-reagent (Sigma Aldrich).
|
| Label |
Cy3
|
| Label protocol |
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) purification. Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium).
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Mouse GE v2 microarray (8X60K, Design 074809 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) and Wash buffer 2 (Agilent Technologies, 37°C, 1 min).
|
| Scan protocol |
Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software
|
| Description |
3
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent Technologies, Santa Clara, CA) using default parameters (protocol GE1_1010_Sep10 and Grid: 074809_D_F_20150624). 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 minimal weight of 1 for 38 out of 47 microarrays or with a minimal weight of 4 per group from at least one experimental group. At this step, 39696 spots out of 62976 were selected. The mean signal of the 31 first samples and 16 last ones were substracted to individuals signals to correct the batch effect of the 2 serials (characteristics:WashBatch) observed during the microarray washing procedure. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore R 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 37027 rows each corresponding to a unique ProbeName (provided as data Matrix).
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| |
|
| Submission date |
Oct 06, 2020 |
| Last update date |
Oct 31, 2022 |
| Contact name |
Hervé Guillou |
| E-mail(s) |
[email protected]
|
| Phone |
0582066389
|
| Organization name |
INRAE
|
| Department |
ToxAlim
|
| Lab |
TIM
|
| Street address |
180 Chemin de Tournefeuille
|
| City |
Toulouse |
| ZIP/Postal code |
31027 |
| Country |
France |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE159086 |
Effect of pemafibrate on liver gene expression in wild-type mice and in mice with hepatocyte-restricted deletion of PPARalpha |
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