Whole-Transcriptome Profiling by Microarray

Total RNA was isolated by using RNeasy MICROKit (Qiagen GmbH, Hilden, Germany) following manufacturer’s recommendations. Total RNA concentration and quality control was assessed using RNA 6000 pico kit (Agilent, Santa Clara, CA, USA). Due to the small amount of RNA obtained, RNA samples were amplified by using Ovation PicoSL System V2 (NuGene Technologies, CA, USA) and ENCORe Biotin module (NuGene technologies). RNA MinElute Reaction Cleanup Kit (Qiagen) was used to purify amplified RNA. RNA samples were labeled and hybridized by using GeneChip Hybridization Control Kit and GeneChip Hybridization, Wash and stain Kit HT (Affymetrix, Santa Clara, CA, USA), respectively. Labeled RNA samples were then hybridized to Affymetrix HG-U219 genechips (16 arrays plate) (Affymetrix) according to the manufacturer’s instructions. Data normalization and analysis was performed using GeneSpring GX12 (Agilent, Santa Clara, CA) as described previously [71 (link)]. For detection of differentially expressed genes, a P-value cut-off of 0.05 was used in combination with a fold-change cut-off of 2.0. Raw data are made publically available on the NCBI Gene Expression Omnibus database, with accession number GSE68000.