3000 7g plus scanner

For DRGs, total RNA was concentrated by speed vacuum to reach ≥100 ng/μL as measured by UV spectrophotometry. Gene expression profiling was performed using 10 Affymetrix GeneChip Rat Gene 1.0 ST Arrays. The array platform interrogates 27,596 genome-wide whole-transcripts using 792,454 distinct well-annotated probes with an average of 29 probes per gene (excluding controls). cDNA was generated from total RNA using the Ambion WT Expression Kit according to the manufacturer’s protocol. The cDNA was fragmented and end labeled following the GeneChip Whole Transcript Terminal Labeling and Hybridization protocol and hybridized to the arrays for 16 h at 45°C in the GeneChip Hybridization Oven 640. Washing and staining with streptavidin-phycoerythrin was performed using the GeneChip Hybridization, Wash, and Stain Kit, and the GeneChip Fluidics Station 450. Chips were scanned using the Affymetrix 3000 7G Plus scanner and the GeneChip Command Console software.

Gene microarray was performed in duplicates and initial analysis was performed by Affymetrix GeneChip Service, LS Sciences, LLC. Full analysis was performed for two samples: HT-29 monolayer cell and HT-29 spheroids. Raw data, metadata, .CEL and .CHP were deposited in the NCBI GEO database, Accession number: GSE65433. Total RNA was isolated using the mirVana™ miRNA Isolation Kit according to the manufacturer's protocol and RNA intergrity was confirmed on the Nanodrop ND-1000 and analyzed with Agilent 2100 bioanalyzer. The samples were amplified and labeled using the Affymetrix 3′IVT labeling kit (Lot NO. 1010021) and hybridized with the GeneChip® Human Genome U133 Plus 2.0 Array for 16 hr. After hybridization, samples were washed and stained with the Affymetrix fluidics station 450. The arrays were scanned with the Affymetrix 3000 7G plus scanner. Dat and Cel files were obtained by the AGCC software, and the CHP files were generated with mas5 method by Affymetrix Expression Console. The data was imported into the Agilent GeneSpring GX software for further analysis. The data obtained through GeneChip® scanning was analyzed using Affymetrix® Microarray Suit Software 5.0. The genes whose expression was significantly altered by 2-fold in either direction were subjected to Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com).