Human mirna oligo chip

SEVs were isolated from conditioned medium of HOS cells or 143B cells using MagCapture Exosome Isolation Kit (FUJIFILM Wako). MiRNAs contained in the SEVs were detected using Human miRNA Oligo chip (Toray Industries Inc., Tokyo, Japan). The value of each miRNA was normalized by correcting the median of the signal intensities to 25, resulting in a global normalization value. Each ratio of miRNA in 143B-SEV to HOS-SEV was calculated by dividing the global normalization value of 143B-SEV by that of HOS-SEV.

MiRNA array analysis was performed as described in our previous study [18 (link)]. Total RNA was extracted from tissue samples using a miRNAeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. MiRNA array was performed using a Human miRNA Oligo Chip (v. 21.0; Toray Industries, Tokyo, Japan). Total miRNA samples (200 ng) were labeled using a miRCURYHy3/Hy5 Power Labeling Kit, slides were scanned in a 3D-Gene Scanner 3000 (Toray Industries), and the fluorescence data were analyzed using 3D-Gene Extraction software (v. 1.2; Toray Industries). Quantile normalization was performed on raw data exceeding the background level, and differentially expressed miRNAs were identified by the Mann–Whitney U test. Hierarchical clustering was performed using the farthest end method, with the absolute non-central Pearson correlation coefficient as the metric. A heat map was created based on the relative expression intensity of each miRNA, using log2 of the fold change.

Total RNA was extracted from KYSE-150 cells (1.0 × 10⁶ cells in a 100-mm dish) treated with or without 100 nM Gal-9 for 6 h using the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA amount was quantified with an RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA) and the samples were labeled using miRCURY Hy3/Hy5 Power labeling kit (Exiqon, Vedbaek, Denmark), followed by the hybridization with a human miRNA Oligo chip (v.21.0; Toray Industries, Tokyo, Japan). The chips were scanned by 3D-Gene Scanner 3000 (Toray Industries). The raw intensity of the image was read using 3D-Gene Extraction Version 1.2 software (Toray Industries). The changes in the miRNA expression between Gal-9–treated and control samples were analyzed with GeneSpring GX v 10.0 (Agilent Technologies). Quantile normalization was performed on the background subtraction data. Differences in miRNA expression were tested by Mann–Whitney U test. Hierarchical clustering was performed using the furthest neighbor method with the absolute uncentered Pearson’s correlation coefficient as a metric. A heat map with relative expression intensity of each miRNA was generated, wherein the base-2 logarithm of the intensity was median-centered for each row.