Plasma samples were collected from EDTA-Na blood samples by centrifugation at 1,500×g and stored at -80°C. RNA was extracted from the plasma samples using the 3D-GeneⓇ RNA extraction reagent from a liquid sample kit (TORAY, Tokyo, Japan), according to the manufacturer's instructions. The extracted total RNA was labeled with the 3D-GeneⓇ miRNA labeling kit (TORAY). Labeled RNAs were hybridized onto 3D-GeneⓇ Human miRNA Oligo chips (TORAY) (12 (link)). The annotation and oligonucleotide sequences of the probes conformed to the miRBase miRNA database (http://www.mirbase.org/ ). After careful washing, the fluorescence signals were scanned with a 3D-GeneⓇ Scanner (TORAY) and were analyzed using the 3D-GeneⓇ Extraction software program (TORAY).
The raw data of each spot were normalized by substitution with the mean intensity of the background signal (determined by the signal intensities of all of the blank spots' and 95% confidence intervals). The measurements of spots with signal intensities that were greater than two standard deviations of the background signal intensity were considered to be valid. The relative expression level of a given miRNA was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments. The normalized data were globally normalized per array, such that the median signal intensity was adjusted to 25.
The raw data of each spot were normalized by substitution with the mean intensity of the background signal (determined by the signal intensities of all of the blank spots' and 95% confidence intervals). The measurements of spots with signal intensities that were greater than two standard deviations of the background signal intensity were considered to be valid. The relative expression level of a given miRNA was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments. The normalized data were globally normalized per array, such that the median signal intensity was adjusted to 25.