Circulating microRNA Quantification Protocol

The seven microRNAs selected during the discovery phase were then measured by RT-qPCR in triplets for each sample and each microRNA using Taqman™ microRNA assays (ThermoFisher, Waltham, MA, USA) (Supplemental Table S4). First, the circulating microRNAs were isolated using the miRNeasy Serum/Plasma Kit (Qiagen, Venlo, Netherlands). Second, microRNAs were reverse-transcribed to cDNA with the Taqman™ MicroRNA Reverse Transcription Kit (ThermoFisher, Waltham, MA, USA). Third, samples were measured in triplicate using RT-qPCR on the CFX384 Touch Real-Time PCR Detection System (Bio Rad, Hercules, USA). Cel-miR-39 was used as a spike-in control to normalize the expression levels of microRNAs, using the delta threshold cycle (∆C_T) method. Three μL of 5 nM cel-miR-39 was added to a total sum of 15 μL of reverse transcription mix [49 (link)]. All microRNAs were measured in a blinded fashion, regarding the case vs. control status of the samples.