To stabilize RNA, 0.4ml of drawn whole blood was mixed with 1.2ml RNAlater (Ambion, Naerum, Denmark) and stored at -20 °C for the first 24 hrs followed by longer term storage at -80 °C until use. Total RNA was extracted according to the manufacturer's instructions.
Label
Cy3
Label protocol
Dye-labeled cRNA (Cy3) was synthesized following the Agilent one-color Quick-Amp labeling protocol (Agilent Technologies, Amstelveen, The Netherlands).
Hybridization protocol
Hybridization according to Agilent instructions on 4x44K human whole genome microarrays.
Scan protocol
Slides were scanned on a GenePix® 4000B Microarray Scanner (Molecular Devices, Sunnyvale, USA). Cy3 was excited at a wavelengths of 532 nm. Laser power was set to 100%. The photo multiplier tube gain was set to a saturation tolerance of 0.02% to minimize background and saturated spots. Photomultiplier gain was set to 543 (Cy3), based on automatic establishment of ideal scan settings. Images were subsequently saved (resolution 5 micron, 16 bit tiff image).
Description
Non-smoker
Data processing
Data preparations were performed in GenePix Pro software (version 6.0, Molecular Devices) and included in chronological order: background subtraction, omission of bad spots (controls and irregularly shaped spots), and flagging of spots (control spots and bad spots). One raw data file was created by GenePix Pro (a GPR file). Quantile normalization and log (base=2) transformations of spot intensities were calculated in ArrayTrack (version 3.4).
