miRNA Array Analysis

Quantitative real-time polymerase chain reaction (qRT-PCR)-based high-throughput miRNA profiling was performed at QuantoBio Biotechnology Co. Ltd. (Beijing, China). Briefly, total RNA extracted from peripheral blood mononuclear cells (PBMCs) was isolated using TRIzol® (Invitrogen, Carlsbad, USA). Escherichia coli poly (A) polymerase was used to add adenines to the 3′ end of RNA molecules lacking a poly (A) tail. After oligo dT annealing, a universal tag was attached to the 3′ end of cDNAs during cDNA synthesis using retrotranscriptase Superscript III (Invitrogen). With this universal tag, a SYBR®-based qRT-PCR was performed using miRNA-specific forward primers and a reverse universal primer mix. Of note, U1 and U6 were used in the training cohort for normalization. The variation of change in the threshold cycle (CT, target-CT, and control) was evaluated and used as a relative qualitative value.