RT-qPCR
To validate the results of RNA-seq analyses, cDNA was prepared from 15 samples from AS patients and control donors via reverse transcription. The levels of three up-regulated and three down-regulated DECs were then assessed in these samples via qPCR using the Universal SYBR Green Master Mix. The primers that had been designed and synthesized by OG-Biotech Inc. (Table 1). Relative circRNA expression was assessed via the 2−ΔΔCq approach, and receiver operating characteristic (ROC) curve analyses were used to differentiate between samples in the control and AS patient groups.
Table1: Specifific circRNAs primers used for qRT-PCR analysis