Animals were 6 weeks old at the begining of the study. Animals had ad libitum access to water and feed for the duration of the trial. The first group of pigs was fed with the control diet while the second group was fed with control diet artificially contaminated with 10 ppm Fumonisin B1. They were fed during 4 weeks prior to sacrifice. They were cared for in accordance with the National Institute of Health Guide and the French Ministry of Agriculture for the Care and Use of Laboratory Animals. At the conclusion of the experiment, immediately after electrical stunning, the piglets were sacrificed by exsanguination. A portion of the jejunum, liver, spleen and jejunum with Jejunal Peyers' patches were collected from euthanatized animals, flash-frozen in liquid nitrogen, and stored at -80°C until processed for total RNA extraction.
Growth protocol
12 crossbred weanling piglets housed in floored indoor pens were used in this study. The piglets were weaned at the age of 28 days and transported to the animal facility of the INRA Research Center in Food Toxicology (Toxalim, Toulouse, France).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted in lysing matrix D tubes (MP Biomedicals, Illkirch, France) containing guanidine thiocyanate-acid phenol (Eurobio, Courtaboeuf, France). Quality of these samples was assessed on Bioanalyzer 2100 (Agilent RNA 6000 Nano Kit, Agilent Technologies, Santa Clara, CA). All samples had a RIN over 7.5.
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 manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to GPL19893 microarray (Agilent 8X60K, Design 037880) 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
re-analysis of GSM2578909
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: 037880_D_F_20120213). 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, 45693 spots out of 61625 were selected. Data were then stored in an ExpressionSet object and normalized by the qsmooth method from the qsmooth R library, considering tissues factor levels to normalize by tissue. 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 45692 rows each corresponding to a unique ProbeName (provided as data Matrix).
