2.5.4. miRNA Analysis with Microarray

The miRNA expression profile was analyzed using a microarray (Gen-Chip miRNA 4.0 array, Affymetrix Inc., Santa Clara, CA, USA). The data were compared using three different methods (ExoCAS-2, exoEasy, and UC). Total RNA (130 ng), which included tissue miRNA, was labeled with biotin using FlashTag. Samples labeled with the TM Biotin HSR RNA Labeling Kit (Affymetrix) were mixed into the Affymetrix miRNA micro-array using the GeneChip^® Hybridization Oven as directed by the manufacturer. The labeled RNA was heated to 99 °C for 5 min and subsequently to 45 °C for 5 min. RNA-array hybridization was performed using the Affymetrix^® 450 Fluidics station for 16 h with stirring at 48 °C at 60 rpm. The chips were cleaned and stained using the Genechip Fluids Station 450 (Affymetrix). Each chip was scanned using the Affymetrix GCS 3000 scanner (Affymetrix). The signal value was determined using the Affymetrix^® GeneChip^® Command Console software (Affymetrix Inc., Santa Clara, CA, USA).