Genespring gx software 14 9

For the microarray analysis, total RNA (miRNA) was isolated from the Neuro2a cells using Triagent (Molecular Research Center, Cincinnati, OH, USA), and the quality was evaluated by a 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). Cy3-labeled probes were prepared from 200 ng of total RNA using the Low Input Quick Amp Labeling Kit, one-color, (Agilent Technologies, #5190-2305) and hybridized with a microarray slide (Whole Mouse Genome DNA microarray 4x44K Ver. 2.0; Agilent Technologies) for 17 h at 65 °C. The microarray slide was washed and scanned with a microarray scanner (ArrayScan, Agilent Technologies) to obtain the fluorescent signal of the probes. The signal was processed for digitization using Feature Extraction software 10.7 (Agilent Technologies) and analyzed with GeneSpring GX software 14. 9 (Agilent Technologies) for gene expression.

RNA was extracted from cells cultured in a monolayer 3 days after cell passage and from spheroids 3 days after the start of culture using SphereRing. For the DNA microarray analysis, tcDNAs were prepared and applied to the Human Gene ST 2.0 microarray (Affymetrix, Thermo Fisher Scientific, Cleveland, OH, USA) according to the manufacturer’s instructions. The GeneChip WT PLUS Reagent Kit (Affymetrix) was used to synthesize cDNA samples to be hybridized to the microarray probes. For hybridization, we used GeneChip Hybridization Oven 645 (Affymetrix). After hybridization, the microarrays were washed and reacted in a GeneChip Fluidics Station 450 (Affymetrix) using the GeneChip Hybridization, Wash, and Stain Kit (Affymetrix) to colorize them according to the amount of the combined cDNA. The hybridized cDNAs were measured and quantified by a GeneChip Scanner 3000 7G. Data were analyzed using Genespring 14.9.1 (Agilent) and Ingenuity Pathway Analysis (IPA, Qiagen) [43 (link)]. Up- or downregulated genes were extracted using the GeneSpring GX software 14.9 (Agilent). IPA (Qiagen, Hilden, Germany) was used for gene network evaluation [45 (link),46 (link)].