CircRNA microarray analysis
Three pairs of human OS and corresponding adjacent nontumor tissue samples were used for the circRNA microarray assay to determine differentially expressed circRNAs. The microarray hybridization was performed based on the manufacturer’s standard protocols (Agilent Technologies, USA), which included purifying the RNA, transcribing it into fluorescent cRNA, and then hybridizing it onto the Human circRNA Arrays (Agilent Technologies, USA). Finally, the hybridized slides were washed, fixed and scanned to images by an Agilent Scanner G2505C. The data collection was performed using Agilent Feature Extraction software (version 11.0.1.1). The raw data were quantile normalized, and further data analysis was performed with the R software package, GeneSpring GX (Agilent Technologies, USA) and gene expression dynamics inspector (GEDI). The statistical significance of differentially regulated circRNAs between OS tissue (T) and adjacent nontumor tissue (N) was identified through p-values and fold changes. Significantly differentially expressed transcripts were retained by screening for a fold change ≥ 2.0 and P < 0.05. Hierarchical clustering was performed to generate an overview of the characteristics of expression profiles based on the values of all expressed transcripts and significant differentially expressed transcripts.