2.3. Retrotranscription and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

A volume of 9.16 µL of RNA from plasma was retrotranscribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. The reaction mixture was incubated at 16 °C for 30 min, at 42 °C for 30 min, and at 85 °C for 5 min in a thermal cycler. To determine miRNA expression levels, qRT-PCR was performed. A total of 2 µL of cDNA was amplified with 5 µL of Xpert Fast Probe 2× MasterMix (GRiSP, Portugal), 0.5 µL of Taqman microRNA assays (Assay ID 000602 and ID 000435 for miR-30b-5p and miR-99a-5p, respectively, Thermo Fisher Scientific, Waltham, MA, USA) and 2.5 µL of nuclease-free water. qRT-PCR reaction was carried out on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) under the following conditions: 98 °C for 3 min, 45 cycles of 95 °C for 10 s, 60 °C for 30 s, and 37 °C for 30 s. RNU38B (assay ID 001004 Thermo Fisher Scientific, Waltham, MA, USA) was employed to normalize the expression of miRNAs. A standard curve of four serial 10-fold dilutions of cDNA was run in each plate and used to calculate the expression of miRNAs. All samples were analyzed in triplicate.