|
| Status |
Public on Oct 13, 2017 |
| Title |
Liver Fasted 1 |
| Sample type |
RNA |
| |
|
| Source name |
Fasted for 24 hours
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6J
gender: male
genotype: wild-type
tissue: liver
|
| Treatment protocol |
None
|
| Growth protocol |
Mice were housed since birth in 12h light/12h dark period and fed a Safe 04 chow diet.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol reagent according to the manufacturer's instructions. RNA amounts were determined by NanoDrop®ND-1000 and the RNA quality was assessed using RNA 6000 NanoChips with the Agilent 2100 Bioanalyzer.
|
| 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) 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 manufacturers instructions. 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 v2microarray (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 |
s19
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) 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 3 per group from at least one experimental group. At this step, 38622 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 35967 rows each corresponding to a unique ProbeName (provided as data Matrix).
|
| |
|
| Submission date |
Dec 16, 2016 |
| Last update date |
Oct 13, 2017 |
| 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) |
| GSE92502 |
Liver transcriptomic response to fasting, high glucose or a high fructose challenges in wild-type C57Bl/6J male mice |
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