Total RNA from 400 µL of serum was extracted using TRIzol™ reagent (Invitrogen Corporation, Carlsbad, CA, USA) based on TRI Reagentâ protocol, eluted in 10 µL of RNase-free water, and stored at −80°C until analysis. The total RNA concentration was determined using a NanoDrop ND-200 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the quality of RNA was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Serum levels of miR-23a (cat. no. 4427975; Applied Biosystems, Carlsbad, CA, USA) and miR-181b (cat. no. 440886; Applied Biosystems) were evaluated by qRT-PCR with the use of the TaqMan™ Advanced miRNA cDNA Synthesis Kit (cat. no. A28007; Applied Biosystems), TaqMan™ Fast Advanced Master Mix (cat. no. 4444557; Applied Biosystems), and specific TaqMan Primers. U6 (RNU6B; cat. no. 4427975; Applied Biosystems) was used for normalization of the data based on 2−ΔΔCT method as described previously [28 (link)].
Serum levels of miR-23a (cat. no. 4427975; Applied Biosystems, Carlsbad, CA, USA) and miR-181b (cat. no. 440886; Applied Biosystems) were evaluated by qRT-PCR with the use of the TaqMan™ Advanced miRNA cDNA Synthesis Kit (cat. no. A28007; Applied Biosystems), TaqMan™ Fast Advanced Master Mix (cat. no. 4444557; Applied Biosystems), and specific TaqMan Primers. U6 (RNU6B; cat. no. 4427975; Applied Biosystems) was used for normalization of the data based on 2−ΔΔCT method as described previously [28 (link)].