Rat Transcriptome Analysis of CGC Treatments

Total RNA was extracted from CGC treated with vehicle, CGC treated with candesartan, CGC treated with glutamate and CGC treated with candesartan and glutamate. Each group consisted of five independent experiments. Standard procedures for labeling, hybridization, washing and staining were as per manufacturer’s recommendation (Affymetrix, Santa Clara, CA, USA). Briefly, the RNA was purified using a RiboPure Kit (Ambion, Austin, TX, USA) according to the manufacturer’s protocol. The quality and quantity of RNA were ensured using the Bioanalyzer (Agilent, Santa Clara, CA, USA) and NanoDrop (Thermo Scientific, Waltham, MA, USA), respectively. For RNA labeling, total RNA (300 ng) was used in conjunction with the Affymetrix-recommended protocol with the WT Plus Reagent Kit catalog #902280. The hybridization cocktail containing the fragmented and labeled complementary DNAs (cDNAs) was hybridized to the Rat GeneChip 2.0 ST chips. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols from Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Molecular Probes, Carlsbad, CA, USA) and enhanced by using an antibody solution containing 0.5 mg/ml biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA, USA). The probe arrays were scanned using an Affymetrix Gene Chip Scanner 3000. Gene expression intensities were calculated using the Gene Chip Operating software 1.2 (Affymetrix). A GC-corrected robust multichip analysis (RMA) normalization model was used to correct for background, and nonspecific binding. All analyses were performed using Partek Genomics Suite (Fig. 1). The raw data is submitted to Gene Expression Omnibus (GEO) under accession GSE67036.