RNeasy mini-kit (Qiagen, Chatsworth, CA) extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA.
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on the Mouse Genome MG-U74A, B, C Probe GeneChip array using Affymetrix Hybridization Oven 640. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using Affymetrix GeneChip Scanner 3000.
Description
no additional information
Data processing
GeneChip software was used to determine the average difference (AD) in the levels of gene expression among genes on the array. The mean AD for 3’-terminal probe sets corresponding to four constitutively expressed genes (beta-actin, GAPDH, pyruvate carboxylase and transferrin receptor) were calculated, and hereafter we use STD to refer to the mean AD of the control probe set. To normalize staining intensity among chips, the AD values for all genes on a given chip were divided by the ratio of the STD for each chip to the average STD for all chips. Normalized AD values less than 0.1 were set to 0.1. Then, the dataset was sorted by the ratio of the mean AD of each gene in a target group to that in a reference group in order to identify highly expressed genes from within an experimental group.
