Mirna cdna synthesis kit with poly a polymerase tailing

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The reverse transcription of mRNA was performed using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Tokyo, Japan). Quantitative real-time PCR was performed using a standard protocol from the SYBR Green PCR kit (Toyobo, Osaka, Japan) on a Biorad-CFX96 thermal cycler (Hercules, USA). GAPDH was used as an internal control. Each sample was analyzed in triplicate. Primers specific to miR-125b-5p and RNU6B were purchased from TsingKe (Beijing, China). For miRNA detectio, 1 μg of total RNA was reverse transcribed using a miRNA cDNA Synthesis Kit with Poly(A) Polymerase Tailing (ABM, Peterborough, Canada). Data were analysed according to the 2^-ΔCq (Comparative Cq) method. The primers used in this study are presented in Additional file 1: Table S1.

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.