Total RNA was extracted from three pools of each endometrial sample (CDd7, CDd14, NPd7 or NPd14 ) using RNeasy mini kit (Qiagen). RNA amplification was perfomed using MEGAscript® T7 Kit (Ambion, Inc., Austin, TX, USA) according to GeneChip® expression analysis technical manual (P/N 702232)
Label
biotin
Label protocol
The cRNA was biotin labeled using the GeneChip® IVT Labeling Kit (Affymetrix, Inc., Santa Clara, CA, USA) according to GeneChip® expression analysis technical manual (P/N 702232)
Hybridization protocol
The biotin-labeled cRNA was fragmented. Following this, a hybridization cocktail consisting of fragmented and labeled cRNA, control oligonucleotide B2 (3nM), 20X eukaryotic hybridization controls (bioB, bioC, bioD, cre), 2X hybridization mix, DMSO and RNAse free water were mixed to a final volume of 200 µl and heated to 99°C for 5 followed by warming at 45°C for 5 min. The samples were then hybridized to GeneChip® Bovine Genome Array for 16 h at 45°C in hybridization oven. For each sample of CDd7, CDd14, NPd7 or NPd14, three biotin labeled cRNA hybridizations were performed.
Scan protocol
The arrays were washed and stained using Fluidics Station 450/250 and scanned using the GeneChip® scanner 3000 integrated with Affymetrix® Microarray Suite software as recommended in GeneChip® expression wash, stain and scan user manual (P/N 702731).
Data processing
The microarray data normalization and background correction was performed using guanine cytosine robust multi-array analysis (gcrma) . The quality of the arrays after hybridization was evaluated by assessing the absent and present calls of the control probesets. Differentially expressed genes were generated using linear models for microarray data analysis (LIMMA). For this, we used R software, (www.r-project.org) and bioconductor packages (www.bioconductor.org).
