miRNA labeling and array hybridization.

After RNA isolation from the samples, the miRCURY Hy3 power labeling kit (Exiqon, Vedbaek, Denmark) was used for the miRNA labeling of the two pooled total RNA groups according to the manufacturer's guidelines. Briefly, 1 μg of each sample was labeled using T4 RNA ligase in calf intestinal alkaline phosphatase (CIP) buffer and CIP (Exiqon). After stopping the labeling procedure, the Hy3-labeled samples were hybridized at 56°C overnight on the miRCURY LNA array (v.16.0) (Exiqon), containing probes for 1,223 human miRNAs, in a 12-bay hybridization system (NimbleGen Inc., Madison, WI). Following hybridization, the slides were washed several times using a wash buffer kit (Exiqon) and finally dried. Each miRNA spot was replicated four times on the same slide, and two microarray chips were used for each group.