|
| Status |
Public on Sep 15, 2022 |
| Title |
Liver-LWT_Beta3-Rep6 |
| Sample type |
RNA |
| |
|
| Source name |
Liver
|
| Organism |
Mus musculus |
| Characteristics |
genotype: LWT
treatment: Beta3
tissue: Liver
|
| Treatment protocol |
All mice were housed at 21-23°C on a 12-hour light (ZT0-ZT12) 12-hour dark (ZT12-ZT24) cycle and had free access to the standard rodent diet (Safe 04 U8220G10R) and tap water. ZT stands for Zeitgeber time; ZT0 is defined as the time when the lights are turned on. All mice used in this study were males and were sacrificed at ZT16.
|
| Growth protocol |
Mice were fed ad libitum a standard rodent diet. For treatment with beta3 agonist receptor agonist: CL316243 (3 mg/kg body weight; Sigma Aldrich) in vehicle (0.5% carboxymethyl cellulose in sterilized water) was given by gavage at ZT10. All mice were sacrificed at ZT16.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was extracted from liver with TRIzol reagent (Invitrogen).
|
| 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).
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| |
|
| 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
|
| 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 20 out of 24 microarrays or with a minimal weight of 4 per group from at least one experimental group. At this step, 33998 spots out of 62976 were selected. 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 31367 rows each corresponding to a unique ProbeName (provided as data Matrix).
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| |
|
| Submission date |
Jan 26, 2021 |
| Last update date |
Sep 15, 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) |
| GSE165558 |
Effect of beta3 adrenergic activation on liver gene expression profile in mice (C57Bl/6J) expressing hepatocyte PPARalpha or not |
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