Agilent human mirna array v19

The total RNAs were dephosphorylated by phosphatase and incubated with the Labeling Spike-In kit (Agilent Technologies Inc.) at 37°C for 30 min. The dephosphorylated RNAs were denatured by dimethyl sulfoxide (DMSO) and subsequently incubated at 16°C in a circulating water-bath or cool block for 2 h. The labeled RNAs were purified with spin columns to remove DMSO in the samples, dried in vacuum concentrators at 45–55°C for 1 h and dissolved in nuclease-free water. The dissolved RNAs were mixed with Hyb Spike-In solution (Agilent Technologies Inc.) to assemble the hybridization mixture. The mixture was hybridized to the Agilent Human miRNA array V19.0 (Agilent Technologies Inc.), which covers 2,006 human miRNAs, at 55°C for 20 h. The arrays were washed with the Gene Expression Wash Buffer kit and subsequently scanned by the Agilent Microarray Scanner (both from Agilent Technologies Inc.). Data on miRNA microarray images were extracted by Feature Extraction software 10.7.1.1 and normalized by Gene Spring software 12.6 (both from Agilent Technologies, Inc.). The similarity between the samples was analyzed by the principal component analysis (PCA) and correlation plot.