|
| Status |
Public on Feb 01, 2016 |
| Title |
Liver_Control_2 |
| Sample type |
RNA |
| |
|
| Source name |
Liver, Control, replicate 2
|
| Organism |
Rattus norvegicus |
| Characteristics |
gender: female
strain: Wistar
age: 10 days
tissue: liver
treatment: control
|
| Treatment protocol |
At 10 days post-partum female Wistar rats from at least 3 different litters were separated from their mothers for 4 hours (MS=maternal separation) or not (CT=controls). The animals were put back with their mothers for 24h prior to microarray analysis of their livers.
|
| Growth protocol |
Litters of Wistar rats and their mothers were housed at 23+/-1°C on a 12 hour light/dark cycle. 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, liver was removed and an approx. 100mg fragment was 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 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 200 ng of total RNA using the One-color Low Input Quick Amp Labeling kit (Agilent Technologies) 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 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 Rat Genome Microarray 4x44K v3 (design 028282) 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 an Agilent High Resolution C Scanner (G2565CA)
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10 (Agilent) using default parameters (protocol GE1_1010_Sep10 and Grid: 028282_D_F_20110906). 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 at least 8 out of 9 microarrays or with a weight of 1 in 4 microarrays from at least one experimental group. The filtered data were then stored as 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 17604 rows each corresponding to a unique ProbeName (provided as data Matrix).
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| |
|
| Submission date |
Feb 11, 2013 |
| Last update date |
Feb 01, 2016 |
| 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 |
GPL14746 |
| Series (1) |
| GSE44241 |
A short-term maternal separation in early neonate rats markedly increases intestinal permeability, induces bacterial translocation to systemic organs and impacts gene expression in the liver |
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