Plasma miRNA Isolation and Quantification

RNA isolation and RT-qPCR were performed as described previously^{42 (link)}. The total RNA, including miRNAs, was extracted from 100 µL plasma using a 1-step phenol/chloro form purification protocol. To control for variability in the RNA extraction and purification procedures, all samples from a given subject were handled in the same batch. Hydrolysis probe-based RT-qPCR was carried out using a TaqMan PCR kit and an Applied Biosystems 7300 Sequence Detection System^{44 (link)}. The Ct values were determined using default threshold settings, and the average Ct value was calculated from triplicate PCRs. Ct values were normalized to the Let-7d/g/i trio, and the fold change of individual miRNA was determined using the 2^−ΔΔCt equation. The ΔCt was calculated by subtracting the Ct values of the Let-7d/g/i trio from the mean Ct values of the target miRNAs. The ΔCt values were then compared (ΔΔCt) with each participant’s own resting baseline value at the Pre time point (normalized to a fold change of 1).