Quantification of Liver miR-33 Expression

Total RNA was isolated from the liver tissues using Ribo Ex (Geneall Biotechnology Co., Ltd., Korea). Complementary DNA (cDNA) was synthesized from 4 μg of isolated RNA using a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Bioneer Co., Korea). An AccuPower 2X Greenstar qPCR MasterMix (-ROX Dye) (Bioneer Co., Korea) and a fluorometric thermal cycler (Corbett Research, Australia) were used for quantitative real-time polymerase chain reaction (qRT-PCR). Primers used for qRT-PCR are described in the supplementary Table 1. β-actin was used as a reference gene for normalization and the results were relatively quantified using the ΔΔCt method (24 (link)) and expressed as a fold-difference compared with the HC group.
The miR-33 expression was measured as demonstrated before (25 (link)). cDNA was synthesized by using a miRNA cDNA Synthesis Kit with Poly (A) Polymerase Tailing (ABM Inc., Canada) and amplified using the EvaGreen miRNA qPCR Master Mix (ABM Inc.). The miR-33 expression was normalized to U6 snRNA using the 2^−ΔΔCt method.