Quantifying Circulating miRNA Levels

All samples and no-template controls for each assay were run in duplicate. Cycle quantification values (Cq) were excluded from analysis if they did not meet the quality control criteria in QuantStudio 7 Flex Software v.2.1 (Thermo Fisher) or if they were identified as an outlier. To obtain serum levels of the miRNAs of interest, the ΔCt approach (2−ΔCt algorithm) was used. The results are presented as the average of relative values ± SD, calculated as 2 − (Ct of miRNA of interest − Ct of normalizer). From the available approaches for normalization, exogenous reference cel-miR-39 was used as a normalizer for circulating miRNAs [39 (link),40 (link)].