Serum miRNA Profiling by RT-qPCR

Once miRNAs had been extracted from all serum samples, the four selected miRNAs (Hsa-miR-497-5p, Hsa-miR-155-5p, Hsa-miR-423-5p and Hsa-miR-365-3p), as well as the housekeeping (Hsa-miR-93-5p) and Cel-miR-39, were determined using RT-qPCR. Hsa-miR-93-5p was chosen as housekeeping for its widely regarded potential as a reference gene^{21 (link),22 (link)} and because in our previous study it was revealed to be a reference miRNA candidate by the GeNorm algorithm in the profiling stage for our sample pool^{13 (link)}. We used 2 μl of each miRNA sample for retro-transcription (RT) in 6 μl of total volume reaction using the TaqMan Micro RNA Reverse Transcription kit (ThermoFisher) and specific primers for each miRNA contained in the TaqMan miRNA Assay kit (ThermoFisher). Amplification of the RT product was performed using TaqMan Universal PCR Master Mix II (ThermoFisher) and the specific primers and probe for each miRNA present in the TaqMan miRNA Assay kit (ThermoFisher). The reaction was carried out using QuantStudio 5 Real-Time PCR System (ThermoFisher) in 384-well plates, and amplification curves were analyzed with QuantStudio Design and Analysis Software (v1.5.1; ThermoFisher).