Mircury lnatm universal cdna synthesis kit

Total RNA was extracted using Tri-Reagent (Molecular Research Inc., Cincinnati, OH, United States). For miRNA, the miRCURY LNA^TM Universal cDNA Synthesis kit (Exiqon Inc., Woburn, MA, United States) was used for reverse transcription of miRNA. The primers were purchased from Exiqon. The miRCURY LNA microRNA PCR SYBR Green master mix (Exiqon Inc.) was used for qPCR with the following cycle parameters: 95°C for 10 min and 40 cycles at 95°C for 10 s and 60°C for 60 s. Expression of individual microRNA was standardized to the mouse U6 gene (tissue) or miR103 (serum) (Hu et al., 2015 (link); Su et al., 2017 (link)). For mRNA we used a Thermoscript RT-PCR kit (Invitrogen, Carlsbad, CA, United States). Real-time qPCR was performed with SYBR Green PCR Reagents (Bio-Rad, Hercules, CA, United States) using the following cycle parameters: 94°C for 2 min and 40 cycles at 94°C for 15 s, 55°C for 30 s, 72°C for 30 s with final extension at 72°C for 10 min. The quantification cycle (Cq) values were defined as the number of cycles required for the fluorescence signal to exceed the detection threshold. Individual miRNA or mRNA expression was calculated as the difference between the threshold values of the two genes (2-Δcq). Melting curve analysis was routinely performed to verify the specificity of the reaction. Let-7-5p (YP00204767) was ordered from Qiagen (Germantown, MD, United States).