Microrray hybridization and data analysis
Total RNA was prepared using a commercial kit (Qiagen, Valancia, CA, USA) according to the manufacturer's instructions. Gene expression profiles were obtained using the CodeLink Uniset Mouse Expression Bioarray (Amersham, Buckinghamshire, UK). The 30,000 gene probes contained in the bioarray allows the detection of differences in gene expression as low as 1.3-fold with 95% confidence. Ten micrograms of total RNA was amplified, and labeled cDNA was produced. Biotin-labeled cDNA was hybridized to the array overnight in a shaking incubator at 37℃, and excess target sequences were washed away using a series of saline sodium citrate washes. The array was stained by treatment with streptavidin-Alexaflour 647 (Molecular Probes, Eugene, OR, USA), the excess was washed away, and the array was scanned at an excitation wavelength of 632 nm using a GenePix scanner (Molecular Devices, Sunnyvale, CA, USA). The resulting image was quantified, and the intensity of each spot was divided by the median spot intensity to provide a scaled and comparable number across multiple arrays.