RNA extraction, DNase treatment and column purification

For RNA extraction, 100 mg of liver tissue was disrupted and homogenized in 800 μL of QIAzol-Lysis Reagent (QIAGEN, Germantown, MD, USA) for 2 min at 20 Hz using a TissueLyzerII with 5 mm stainless steel beads (QIAGEN, Mississauga, ON, Canada) according to the manufacturer’s instructions. To remove lipid/protein contamination, we performed an additional precipitation step according to the protocol of Xu et al. [123] with minor changes. Briefly, crude RNA samples (100 μg) were mixed with an equal volume of Acid-Phenol:Chloroform (5:1 solution, pH 4.5, Ambion/Life Technologies, Waltham, MA, USA) and centrifuged at 17,000 g and 7 °C for 20 min. Then, 190 μL of the aqueous phase was precipitated with 0.1 volumes of 3 M sodium acetate (pH 5.5; Invitrogen/Thermo Fisher, Vilnius, Lithuania) and 2.2 volumes of ice-cold 100% ethanol (Greenfield Global, Brampton, ON, Canada) at − 80 °C for 12 h, and centrifuged at 17,000 g and 7 °C for 30 min. The RNA pellets were then washed in 1 ml of 70% ethanol, centrifuged at 17,000 g and 7 °C for 20 min, air-dried at room temperature for 10 min, and dissolved in 100 μL of nuclease-free water (Invitrogen/Life Technologies, Grand Island, NY, USA) at 55 °C for 5 min. To remove any remaining genomic DNA, 40 μg of precipitated RNA was incubated for 10 min with 6.8 Kunitz units of DNase I (RNase-Free DNase Set, QIAGEN, Mississauga, ON, Canada) and 1 × of the manufacturer’s buffer. DNase-treated RNA samples were then column-purified using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s guidelines. RNA integrity was tested with gel electrophoresis (1% agarose) and RNA purity and yields were measured by A260/280 and A260/230 NanoDrop UV spectrophotometry (NanoDrop, Wilmington, DE, USA). All column-purified samples had A260/280 ratios between 2.0 and 2.2 and A260/230 ratios between 1.9 and 2.3.