Total RNA was extracted from serum samples using the RiboEXTMLS kit (GeneAll, Seoul, Korea). RNA quantity and quality were evaluated using a spectrophotometer (NanoDrop® ND-1000 UV-Vis, Nanogen Inc). RNA quantity and quality were evaluated using a spectrophotometer (NanoDrop® ND-1000 UV-Vis, Nanogen Inc.). Total RNA samples were spiked using the microRNA Spike-In Kit (Agilent Technologies Santa Clara, CA, USA) to assess the labeling and hybridization efficiencies. Labeling and hybridization were performed using the miRNA complete Labeling and Hybridization Kit (Agilent Technologies Santa Clara, CA, USA) according to manufacturer’s instructions. Samples were hybridized to the SurePrint G3 Human miRNA, 8X60K platform (miRBase release 21.0, Agilent Technologies Inc., Santa Clara, CA) containing probes for the detection of 2,549 human miRNAs. Images were scanned using Agilent Microarray Scanner (G2565CA) controlled by Agilent Scan Control 7.0 software. The signal after background subtraction was exported directly into Agilent Feature Extraction Software version 4.0.1.21 (Agilent Technologies, Santa Clara, CA). Normalization was performed using quantile algorithm. MicroRNAs were considered as differentially expressed (DE) if they obtained a p-value<0.05 and a False Discovery Rate (FDR)≤0.05.
Label
Cy5
Label protocol
Total RNA samples were spiked using the microRNA Spike-In Kit (Agilent Technologies Santa Clara, CA, USA) to assess the labeling and hybridization efficiencies. Labeling and hybridization were performed using the miRNA complete Labeling and Hybridization Kit (Agilent Technologies Santa Clara, CA, USA) according to manufacturer’s instructions.
Hybridization protocol
Samples were hybridized to the SurePrint G3 Human miRNA, 8X60K platform (miRBase release 21.0, Agilent Technologies Inc., Santa Clara, CA) containing probes for the detection of 2,549 human miRNAs.
Scan protocol
Images were scanned using Agilent Microarray Scanner (G2565CA) controlled by Agilent Scan Control 7.0 software.
Data processing
The signal after background subtraction was exported directly into Agilent Feature Extraction Software version 4.0.1.21 (Agilent Technologies, Santa Clara, CA). Normalization was performed using quantile algorithm. MicroRNAs were considered as differentially expressed (DE) if they obtained a p-value<0.05 and a False Discovery Rate (FDR)≤0.05.
