Quantifying Circulating miRNA Profiles

The absolute expression (copy numbers) of 575 candidate miRNAs were quantified in each patient and control biospecimen using ID3EAL miRNA Discovery Platform using a miRNA-specific RT-qPCR assay (MiRXES, Singapore) via a controlled workflow described in detail previously (38 (link)). Total RNA from 200 μl of patient serum specimen was isolated using miRNeasy serum/plasma miRNA isolation kit (Qiagen, Germany). Two sets of synthetic spike-in miRNA controls (three each at high, medium, and low concentration) were added to samples before RNA isolation and before RT-qPCR to monitor and normalize technical variations throughout the entire experiment. The absolute expression of each miRNA was normalized using calibrated spike-in controls and determined for each patient’s serum sample (expressed as log 2 copy number/ml serum). The samples were assayed across two plates in this study.