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.