The experimental work on animals was conducted in the Toxalim Unit, INRA, Toulouse (France), followed the European Union guidelines for laboratory animal use and care, and was approved by an independent ethics committee. Eight weeks-old female and male C57 BL/6J mice were purchased from Janvier Laboratories, France. Mean body weights were 18.9 and 23.1g for females and males respectively. The animals were allowed to acclimatise to laboratory conditions for 2 weeks before starting the experiment.
Extracted molecule
total RNA
Extraction protocol
Thirty-six males and 36 females were housed in the laboratory animal room (23 ± 2 ºC) and segregated into 2 groups, each including 18 females and 18 males, fed the pesticides-enriched or the control diet for 52 weeks.
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
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, 34183 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 31532 rows each corresponding to a unique ProbeName (provided as data Matrix).
