Ovation biotin system

Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. The isolated RNA was quantified, and quality was assessed using an Agilent 2100 BioAnalyzer with the RNA Nano Chip (Agilent, Santa Clara, CA, USA). Then, 2 μg of total RNA was used to generate single-stranded antisense cDNA using the Ovation Biotin System (NuGEN Technologies, San Carlos, CA, USA) according to the manufacturer’s instructions. Labeled targets were hybridized to Affymetrix MOE430 2.0 GeneChip microarrays (Affymetrix) for 16 h at 45°C. The arrays were washed and scanned according to standard Affymetrix protocols. GeneSpring 12.0 (Silicon Genetics, Redwood City, CA, USA) was used for data analysis. Microarray data sets have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus under accession number GSE81241.

Total RNA was extracted from cultured cells using a guanidinium isothiocyanate-based buffer containing β-mercaptoethanol and acid phenol [11 ]. Amplification of total RNAs was with the one-cycle Ovation™ biotin system (NuGEN) [17 (link)].