For total RNA isolation, blood sample was collected into PaxGene Blood RNA tubes (PreAnalytiX/QIAGEN Inc., Valensia, CA) before breakfast.
Label
Cy3
Label protocol
100 ng of total RNA was converted to cDNA, amplified, and labeled with Cy3-labeled CTP using the Quick Amp Labeling kit (#5190-0442, Agilent Technologies, Santa Clara, CA) according to manufacturer's protocol.
Hybridization protocol
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent whole human genome 4 × 44 K oligo-DNA microarray (Agilent Technologies, Santa Clara, CA). After hybridization, arrays were washed consecutively by using Gene Expression Wash Pack (#5188-5327, Agilent Technologies, Santa Clara, CA).
Scan protocol
Fluorescence images of the hybridized arrays were generated using the Agilent DNA Microarray Scanner (Agilent Technologies).
Description
Gene expression before breakfast in human blood
Data processing
The feature intensities were extracted with Agilent Feature Extraction software ver.10.7.3.1. The raw microarray intensities were processed by the percentile shift method (75th percentile) with GeneSpring GX 13.0 (Agilent Technologies) so as to normalize the range of expression intensities for inter-microarray. Genes whose expressions were available in more than 50% of hybridizations were included for further analyses. The normalized data were exported from the GeneSpring GX software.
