Transcriptomic Profiling Protocol for Gene and miRNA Expression

RNA isolation, gene and microRNA expression experiments were performed in accordance with our previously published protocols [10 (link), 34 (link)]. In brief, RNA was isolated using Total Tissue RNA Purification Kit from Norgen-Biotek Corp. (Thorold, ON, Canada) and were quantified using NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). Total RNA was labelled and then hybridized to the Agilent Human SurePrint G3 Human GE 8 × 60 k v16 mRNA microarray chip (Agilent Technologies). All microarray experiments were conducted at the Microarray Core Facility (Stem Cell Unit, Department of Anatomy, King Saud University College of Medicine). Data were subsequently normalized and analyzed using GeneSpring 13.0 software (Agilent Technologies). Pathway analyses were conducted using the Single Experiment Pathway analysis feature in GeneSpring 13.0 (Agilent Technologies). Twofold cut-off with P < 0.02 was used. Target prediction was conducted using a built-in feature in GeneSpring 13.0 based on TargetScan database.