Rabbit Microarray Transcriptome Analysis

The eight samples from each group were used as biological replicates for the microarray analysis. RNA from each placental tissue (200 ng) was amplified and labelled with cyanine 3 (Cy3) dye using the One-colour Microarray-based Gene Expression Analysis Low Input Quick Amp Labelling Kit (Agilent Technologies). Excess dye was removed with the RNeasy Mini Kit (Qiagen) and dye incorporation and concentration were determined using the microarray setting on the Nanodrop 2000 and the Bioanalyzer 2100. Hybridization was performed at the CRB GADIE (INRA Jouy-en-Josas, France, http://crb-gadie.inra.fr/). Equal amounts of Cy3-labelled samples (600 ng, Specific Activity > 6.0 pmol Cy3/μg of cRNA) were mixed with blocking agent and fragmentation buffer, and then 24 μl of the mixture was hybridized into the Rabbit Custom Gene Expression Microarrays (GEO accession GPL18913, Agilent-042421 Rabbit BDR version 2, Agilent Technologies) for transcriptomic analysis (12775 genes)^{66 (link)}. After 17 h at 65  °C, the hybridized slides were washed twice for 1 min at room temperature in GE Wash Buffer 1 and 1 min at 37 °C with GE Wash Buffer 2 (Agilent Technologies) and air-dried and scanned immediately.