2.3.12. Isolation and qRT-PCR of Micro-RNAs from Serum

Micro-RNAs from serum samples were isolated using Qiagen miRNeasy Serum/Plasma Kit (Qiagen; Hilden, Germany) following the instructions of the manufacturer. The quality of RNA was determined using NanoDrop2000 (Thermo Fisher Scientific; Hampton, NH, USA). The amount of 10 ng of RNA was reverse transcribed using a TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems; Foster City, CA, USA) and specific primers for miR-21, miR-34a, miR-146a and miR-204 (Applied Biosystems; Foster City, CA, USA). Briefly, for reverse transcription reactions, 10 ng of total RNA was prepared with 3 μL of each of microRNA primers (100 mM), 0.15 μL of 100 mM dNTPs, 1.5 μL of 10X RT buffer, 0.2 μL of 20 units/μL RNase-inhibitor, 1 μL of MultiScribe Reverse Transcriptase (50 units/μL) and 4.15 μL of DEPC treated water in a reaction volume of 15 μL. The RT protocol was as follows: 16 °C for 30 min, 42 °C for 30 min and 85 °C for 5 min. Following reverse transcription, quantitative PCR was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems; Foster City, CA, USA) and oligos from the same assay kits of Applied Biosystems as specified before. Values were normalized with the U6 snRNA as the reference gene (Applied Biosystems; Foster City, CA, USA). According to manufacturer’s guidelines, the relative quantity of each micro-RNA was determined by ∆∆C_T method [58].