fig2

Figure 2. Schematic diagram of the microRNA quantification workflow. (A) Blood collection, plasma isolation, and long-term storage. Peripheral venous blood is collected in BD Vacutainer® EDTA-coated tube and plasma is separated by centrifugation (2,500× g, 15 min, room temperature). Plasma supernatant is removed and centrifuged again and supernatant is aliquoted for storage at -80 °C. (B) Small RNA Isolation from Plasma. microRNA is isolated according to the manufacturer’s instructions of the Qiagen miRNeasy Serum/Plasma Advanced Kit. (C) Small RNA quantification and concentration normalization. The concentration (ng/μL) of eluted microRNA is quantified using the Qubit microRNA assay, according to manufacturer’s instructions. Concentration is subsequently adjusted to 122.55 ng/μL for all samples. (D) Spike-in addition and microRNA specific cDNA generation. miR-39 is exogenously spiked into the microRNA sample, and target-specific cDNA generation is performed utilizing TaqMan microRNA Reverse Transcription Kit following manufacturer’s instructions. miR-39-specific and target-specific cDNA generation are performed separately to avoid cross-reactivity. (E) Droplet generation and microRNA specific PCR amplification. miR-39 cDNA and target-specific cDNA are mixed together with ddPCR supermix (no DUTP), and respective FAM and VIC miR-39 and target-specific cDNA amplification primers. Samples are transferred to Bio-Rad droplet generator microfluidic chips alongside droplet generator oil and transferred to the QX200 Droplet Generator. Droplets are transferred to a deep-well PCR plate, followed by PCR amplification. (F) Droplet reading and data analysis. The samples are read by the Bio-Rad QX200 Droplet Reader and analyzed with Bio-Rad QuantaSoft software. All appropriate instructions in user manuals are adhered to.