circRNAs microarray analysis in colorectal caner tissues

Circular RNA (circRNA) is a kind of transcripts characterized by originating from the back-splicing of pre-mRNA. CircRNAs have higher stability than liner transcripts and are more resistant to RNase R due to their loop structure, which makes circRNAs potential diagnostic markers and therapeutic targets for cancer . The biological functions of circRNAs are widely involved in the proliferation, metastasis, apoptosis, and autophagy of various cancers via multiple ways. To further investage the role of circRNA in CRC, a circRNA micoarray was performed. CapitalBio Technology Human CircRNA Array v2 was designed with four identical arrays per slide (4 x 180K format), with each array containing probes interrogating about 170,340 human circRNAs. Those circRNA target sequences were all from Circbase，Deepbase and Rybak-Wolf20151. Each circRNA was simultaneous detected by a long probe and a short probe. The circRNA array also contain 4974 Agilent control probes.The circRNA array data were analyzed for data summarization, normalization and quality control by using the GeneSpring software V13.0 (Agilent). To select the differentially expressed genes, we used threshold values of ≥2 and ≤−2-fold change and a Benjamini-Hochberg corrected p vlaue of 0.05. The data was Log2 transformed and median centered by genes using the Adjust Data function of CLUSTER 3.0 software then further analyzed with hierarchical clustering with average linkage (Eisen et al., 1998). Finally, we performed tree visualization by using Java Treeview (Stanford University School of Medicine, Stanford, CA, USA).