The blood samples were obtained between 9:00 and 12:00, and then heparinized.
The total RNA was extracted using the QIAmp RNA Blood Mini Kit (QIAGEN K.K., Tokyo, Japan) according to the manufacturer’s instructions. The eluted samples were processed with the standard method of ethanol precipitation, and the RNA pellet was dissolved in RNase-free water. The RNA quantity and quality were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) as recommended.
Total RNA was amplified and labeled with Cyanine 3 (Cy3) using a one-color Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Briefly, 100 ng of total RNA was reverse-transcribed to obtain double-stranded cDNA using a poly dT-T7 promoter primer. The primer, template RNA, and quality-control transcripts of known concentrations and quality were first denatured at 65°C for 10 min and then incubated for 2 h at 40°C with 5× first-strand buffer, 0.1 M dithiotreitol, 10 mM dNTP mix, and AffinityScript RNase Block Mix. The AffinityScript enzyme was then inactivated at 70°C for 15 min. The cDNA products were used as templates for in vitro transcription to generate fluorescent cRNA. The cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3-labeled CTP and then incubated at 40°C for 2 h. The labeled cRNAs were purified using Qiagen’s RNeasy mini spin columns and eluted using 30 µl of nuclease-free water. After cRNA was amplified and labeled, the cRNA quantity and cyanine incorporation were determined using a NanoDrop ND-1000 spectrophotometer and an Agilent Bioanalyzer.
For each hybridization, 600 ng of Cy3-labeled cRNA was fragmented and hybridized at 65°C for 17 h to the Agilent SurePrint G3 Human GE 8×60K v2 Microarray (Design ID: 039494).
After washing, the microarrays were scanned using an Agilent DNA microarray scanner.
The intensity values of each scanned feature were quantified using Agilent feature extraction software version 10.7.3.1, which performs background subtractions. The normalization was performed using Agilent GeneSpring GX version 11.0.2 (per chip: normalization to the 75th percentile shift; per gene: none). The probes that were declared as “present” in all the assayed samples and that displayed a raw intensity value above 50 in at least 2 samples were used for the following statistical analyses.