Microarray analysis reveals potential circRNA biomarkers in human acral lentiginous melanoma

We have completed the Arraystar Human circRNA Array V2 analysis of the 12 samples that you submitted. Total RNA from each sample was quantified using the NanoDrop ND-1000. The sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols. Briefly, total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs. Then, the enriched circular RNAs were amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8x15K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. For the present study, 6 ALM and corresponding paired para-cancer tissues samples stored at the Institute of Dermatology, Chinese Academy of Medical Sciences (Nanjing) were randomly selected from January 2016 to May 2018. RNA was then extracted from these tissues for circRNA microarray analysis. In addition, reverse transcription-quantitative PCR was performed to verify the differentially expressed circRNAs in ALM tissues and melanoma cells including A375, A875 and SK-MEL-28. before a competing endogenous RNA (ceRNA) network constructed by integrating predictions from miRcode, miRanda, and TargetScan tools. Furthermore, gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed for these differentially expressed circRNA. Amongst the 192 differentially expressing circRNAs found, 57 up-regulated and 135 down-regulated circRNAs were found according to the P < 0.05 and |fold change (FC)| > 2 thresholds set. According to RT-qPCR results, it was found that circ_0000569 and circ_0005029 were significantly down-regulated, whilst circ_0000094 was up-regulated in ALM tissue samples compared with paired para-cancer tissues, which was consistent with the microarray analysis results.