2.3. Microarrays

Total RNA samples from five control children and eight epileptic children were analyzed using a microarray according to the manufacturer’s protocol (Two-Color Microarray-Based Gene Expression Analysis/Low Input Quick Amp Labeling; Agilent Technologies, Santa Clara, CA, USA). Briefly, 75 ng of total RNA was converted to cDNA, followed by in vitro transcription and incorporation of Cyanine 3-CTP into the nascent complementary RNA (cRNA), followed by fragmentation and hybridization to Agilent SurePrint Human GE 8 × 60 K Microarrays (Agilent Technologies) for 17 h at 65 °C. The quality control parameters used for cRNA labeling and hybridization were specified by the manufacturers. The microarrays were scanned using a NimbleGen microarray scanner (Roche, Basel, Switzerland), and the signal intensities in the TIFF images were calculated using Feature Extraction software (FE, version 12.0; Agilent Technologies). The microarray data were analyzed, and the associated biological pathways were determined using GeneSpring GX 13.0 software (Agilent Technologies). Differentially expressed genes were selected with a fold change >2.0 and p < 0.05. The Benjamini–Hochberg algorithm was used to compute false discovery rates [23]. The classification of the identified pathways was based on the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG Pathway Maps). The gene ontology analysis for down- and up-regulated genes was submitted to the bioinformatics and functional annotation tool of the Database for Annotation, Visualization and Integrated Discovery (DAVID Bioinformatics Resources) v. 6.8 of the NIAID (National Institute of Allergy and Infectious Disease), NIH (National Institutes of Health).