Genespring software version 14

miRNA profile analysis of the plasma samples was performed using a miRNA Microarray Chip V2.4 (Agilent Technologies, Inc.), which contains probes for 2,549 human miRNAs with a sample input of 100 ng total RNA. Briefly, dephosphorylation was performed by gently mixing the total RNA with 2 µl calf intestinal alkaline phosphatase master mix (Agilent Technologies, Inc.) and incubating the mixture at 37°C in a circulating water bath for 30 min. Subsequently, 2.8 µl 100% dimethyl sulfoxide was added to each sample and incubated at 100°C in a circulating water bath for 5–10 min for denaturation. The samples were labeled using a miRNA Complete Labeling and Hybridization kit and hybridized on an Agilent SureHyb Microarray Hybridization Chamber (both from Agilent Technologies, Inc.). Following hybridization, the chip was washed using GE wash buffer 1 and GE wash buffer 2 (Gene Expression Wash Buffer kit; cat. no. 5188–5327; Agilent Technologies, Inc.). The chip was scanned and the data were extracted using Agilent Feature Extraction Software version 10.7.1.1 (Agilent Technologies, Inc.). Data were standardized using GeneSpring Software version 13.1 (Agilent Technologies, Inc.). Fold change ≥2 and P<0.05 were used to indicate significant differences in gene expression, and cluster analysis was performed using Genespring software version 14.8 (Agilent Technologies, Inc.).