Genechip wt plus reagent kit manual

The quality and quantity of total RNAs were assessed by Agilent bioanalyzer 2100 analysis. Analysis of gene expression was performed using GeneChip^® Mouse Gene 2.0 ST Arrays (Affymetrix, Santa Clara, CA, USA). In situ synthesis of eleven pairs of oligonucleotide probes was conducted for each gene. Single stranded-DNA (ssDNA) was fragmented and labeled from 500 ng of total RNA (GeneChip^® WT PLUS Reagent Kit Manual, Affymetrix). After DNA fragmentation, ssDNA was subjected to hybridization for 16 hours at 45°C (60 rpm; GeneChip^® Mouse Gene 2.0 ST Array). The GeneChips were washed stained, and then scanned using the Affymetrix GeneChip Scanner 3000 7G. The scanned data were converted to intensity values, which were normalized and log transformed.

Fragmented and labeled single-stranded DNA (ss-DNA) was prepared according to the standard Affymetrix protocol from 400 ng total RNA (GeneChip® WT PLUS Reagent Kit Manual, 2001, Affymetrix). Following fragmentation, 3.5 μg of ss-DNA was hybridized for 16 h at 45 °C and 60 rpm on GeneChip® CHO Gene 2.0 ST Array. GeneChips were washed and stained in Affymetrix Fluidics Station 450. GeneChips were scanned using Affymetrix GeneChip Scanner 3000 7G. The data were analyzed by Robust Multichip Analysis using Affymetrix default analysis settings and global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. The normalized and log-transformed intensity values were then analyzed using GeneSpring GX 13.1 (Agilent technologies, CA). Fold-change filters included the requirement that the upregulated genes should be present in ≥ 200% of controls and downregulated genes should be present in < 50% of controls. Hierarchical clustering data were clustered groups that behave similarly across experiments using GeneSpring GX 13.1 (Agilent technologies, CA).