Microarray Analysis of Placental RAS Genes

The microarray analysis (cohort 1) was performed as previously published [28 (link)]. In short, 500 ng of total RNA was converted to double stranded cDNA and this was used to generate biotinylated cRNA probes using the Illumina TotalPrep RNA Amplification Kit. Biotin-labelled cRNA were then hybridized to Illumina RatRef-12 Expression BeadChip (San Diego, CA, USA). Slides were scanned on a BeadStation 500 System using Beadscan software version 3.5.31. No RNA samples were pooled in this analysis, each placental samples was analyzed independently. Samples were hybridized into wells at random. The Array experiment readout was deposited on ArrayExpress and includes gene expression data from microarray and high throughput sequencing studies (ArrayExpress Accession number E-MTAB-1987).
RAS genes from the KEGG Renin Angiotensin System pathway were studied for differential expression using SAM (Microarray Software, Stanford University) analyses, whereby differentially expressed placental genes (i.e. >2 fold expression change) were identified between each of the 4 gestational age groups.