Venous blood (10 ml) was collected from 51 horses using EDTA tubes (BD Vacutainer®). In order to stabilized intracellular RNA, 0.8 ml of venous blood was transferred in a tube containing 0.8 ml of Lysis Buffer DL (Macherey-Nagel, Düren, Germany). After homogenization, the samples were maintained at -20°C until RNA extraction. Total RNA was extracted using the NucleoSpin® RNA Blood kit (Macherey-Nagel, Düren, Germany). The manufacturer’s protocol was modified to obtained sufficient ARN quantities for the Microarray gene expression analyses: extraction was carried out from 1.6 ml of the mix blood and Lysis Buffer DL. Twenty µl of proteinase K was then added to complete blood lysis. To adjust RNA binding conditions, 0.8 ml of 70% ethanol was added. The following steps were not modified, except the elution that was carried out with 40 µl of RNase free water. Total RNA quality was assessed using RNA Pico chips on a Bioanalyser 2100 (Agilent, Boeblingen, Germany) and and its concentration was measured on a Nano Drop One Spectrophotometer (ThermoScientific, Illkirch, France).
Label
Cy3
Label protocol
For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 50 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 Horse_60K_2016_01_22 021322 GE 8x60K (AMID 081421) 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: 081421_D_F_20160126). 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 6 out of 45 microarrays. At this step, 42126 spots out of 62976 were selected. The mean signal of the 16 first samples and 29 last ones were subtracted to individuals signals to correct the batch effect of the 2 serials (characteristics:WashBatch) observed during the microarray washing procedure. 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 30265 rows each corresponding to a unique ProbeName (provided as data Matrix).
