Command console software

We previously described methods for tissue specimen preparation and hybridization [42 (link), 54 (link), 55 (link)]. All processing and analysis of microarrays utilized standard protocols at the University of Miami Microarray Core Facility. Briefly, between 100 to 300 ng of total RNA was reverse transcribed, amplified, then the sense strand cDNA synthesized, labeled, and hybridized on arrays. The amplified, fragmented and biotin-labeled cDNAs were hybridized to the Affymetrix GeneChip Human Gene 2.0 ST microarray according to the manufacturer’s recommendations. Arrays were washed and stained using Affymetrix Fluidic stations 450 and scanned using Affymetrix GeneChip scanner 3000 7G. Image analysis was performed using the Affymetrix Command Console Software (AGCC). Resulting CEL files was imported into Expression Console Software (Affymetrix, Santa Clara, CA, USA) and underwent gene level normalization and signal summarization. The output files from this step were imported in Transcriptome Analysis Console (TAC) 2.0 Software (Affymetrix, Santa Clara, CA, USA) to identify differentially expressed genes and carry out clustering analysis. Only genes with a p-value lower than 0.05 and a fold-change greater than 2 were confirmed by qPCR in a prospective set of 10 NFS and 10 DFS. Microarray profiles are publically available in GEO database under the super series GSE68186.

A GeneChip miRNA 3.0 array (Affymetrix, Santa Clara, CA) was hybridized using 500 ng total RNA per standard Affymetrix protocols. The same RNA preparations used in the mRNA microarray analysis were used for miRNA array analysis. Data extraction was completed using Affymetrix Command Console software. Raw data was analyzed by the following workflow: background detection, RMA global background correlation, quantile normalization, median polish, and log⁡2 transformation with miRNA QC tool software.

Total RNA of the S18 and S26 cell lines was extracted, purified, amplified, and labeled with an Affymetrix WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA, US) to obtain biotin labeled cDNA. The GeneChip Human Transcriptome Array 2.0 array was hybridized and washed using a GeneChip Hybridization, Wash and Stain Kit (Affymetrix), and scanned with an Affymetrix GeneChip Scanner 3000 (Affymetrix). Command Console Software (Affymetrix) was applied to summarize the probe cell intensity data and generalize the CEL file data using default settings. Expression Console was used to normalize the raw data.

Command Console software (version 4.0, Affymetrix, Santa Clara, CA, USA) was used to analyze the array images to obtain the necessary raw data, and then Expression Console software (version 1.4.1, Affymetrix, Santa Clara, CA, USA) was used to perform RNA normalization. To define the expression profiles of each variant, we performed one-way ANOVA with Transcriptome Analysis Console software (version 3.1, Affymetrix, Santa Clara, CA, USA). miRNAs with a p < 0.05 and a |log2(fold change)| > 1 were identified as significantly differentially expressed miRNAs. Hierarchical clustering was performed to show the differences in the expression patterns of these miRNAs among samples.
The target genes of the differentially expressed miRNAs were predicted using the following 5 databases: miRanda, miRDB, miRTarBase, TargetScan, and miRecords. To ensure the accuracy of our prediction, we selected genes enrolled in at least two databases as target genes and subjected them to further analysis.
Finally, KEGG analysis was performed to determine the pathways in which the target genes of these differentially expressed miRNAs are involved. In addition, the target immune function-related pathways of individual miRNAs were predicted using DIANA-miRPath v3.0.

Samples of total RNA from the parental 5637/T24 cell line and 5637/T24 sphere cells were analysed on an Affymetrix HTA 2.0 Array. The arrays were scanned by an Affymetrix GeneChip^® Scanner 3000 (Cat# 00‐00213; Affymetrix, Santa Clara, CA, USA). Command Console Software (Affymetrix) was used with the default settings to control the scanner and summarize probe cell intensity data (CEL file generation). Then, the raw data were normalized by Expression Console.

The array was scanned using Affymetrix Model 3000 G7 scanner and image data was extracted through Affymetrix Command Console software v1.1. The raw.cel file generated through above procedure including expression intensity data was used for the next step. Expression data were generated by Affymetrix Expression Console software version1.4. For normalization, the Robust Multi-Average algorithm implemented in Affymetrix Expression Console software was used.