Genespring gx v10

Total RNA was isolated from CAFs, AHFs, and NFs using the mir-VanaTM miRNA isolation Kit (Ambion, Austin, TX, USA) following the manufacturer’s instructions. Probe synthesis and hybridization to Agilent Human microRNA Microarray v2.0 (Agilent, Santa Clara, CA, USA) were performed by using the miRNA complete labeling and hybridizing Kit (Agilent) following protocols recommended by the producer. Analysis of the arrays was performed using the GeneSpring GX v10.0 (Agilent) and R statistics package (R v2.14.0) as described previously^{15 (link)}.
The mRNA microarray data obtained our previous microarray data of CAFs and NFs. Paired SAM analysis was applied as described previously^{11 (link)}.

OCI-AML2 cells were treated with 10 μM of CLQ or 5 μM of MFQ (All from Sigma-Aldrich) for 24, 30 or 48 hrs before collected for RNA preparation and gene expression profiling analysis according to our previous report [19 (link)]. Microarray data were analyzed using GeneSpring GX v10.0 (Agilent), and lists of genes deregulated > 2-fold were subject to further study.

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.