Liver miRNA Profiling Protocol

Representative liver tissue was stored in RNAlater stabilization solution (Ambion, Austin, TX, USA) at − 80 °C. miRNAs were extracted using the miRVana miRNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer's instructions. The integrity and amount of the RNA fraction highly enriched in sRNA species ≤ 200 nt was determined using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA purity was assessed from the ratio of A260/A280; where 1.8–2.1 is expected^{34 (link)}. Ten ng of sRNAs were used to performed retrotranscription using the Advanced miRNA cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's instructions. Reverse transcription products were diluted to 50 µL, and 2.5 µL of the diluted sample were used for qPCR reactions, with a total volume of 10 µL/Rx and 1X Thermo specific TaqMan probes were used for qPCR for mmu-miR-34a-5p, mmu-miR-122-5p, mmu-miR-21a-5p and mmu-miR-103-3p, in a LigthCycler 96 equipment (Roche Molecular Systems, Pleasanton, CA, USA). qPCR was performed as follows: 1 cycle at 95 °C for 20 s, 40–60 cycles 95 °C for 1 s followed by 60 °C for 20 s. Probes catalog number are shown in Supplemental Table 1. Gene expression was normalized against mmu-miR-16-5p. Analysis was performed using the 2^−ΔCT method^{35 (link)}.