normalization data transformation protocol
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026652_D_F_20110819) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. ID_REF = VALUE = Normalized signal intensity
array scanning protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100%).
1,650 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Human Gene Expression 4x44K v2 Microarrays (G4845A) for 17 hours at 65°C in a hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using a Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy mini column purification (QIAGEN, Valencia, CA).
The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum at 37oC in a humidified incubator with 5% CO2.
nucleic acid extraction protocol
Total RNA was prepared using a High Pure RNA Isolation Kit (Roche Diagnostics K.K., Tokyo, Japan) following the manufacturer's recommendations. RNA was quantified using a NanoDrop ND-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).