Amv retrotranscriptase system

Frozen liver tissue (~50 mg) was cut into pieces, and homogenized in 1 ml of TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using an electric homogenizer (Tissue Tearor, BioSpec Products Inc., Bartlesville, OK, USA) on ice. Total RNA was extracted according to the manufacturer's protocol. cDNA was synthesized from 2.5 µg RNA using random primers and an AMV Retrotranscriptase system (Takara Biotechnology Co., Ltd., Dalian, China) using the following temperature protocol: 30°C for 10 min; 42°C for 30 min and 95°C for 5 min. The SYBR Green RT-qPCR was performed using the ABI StepOne Plus and software (Applied Biosystems; Thermo Fisher Scientific, Inc.). All reactions were performed in triplicate. In a final reaction volume of 20 µl, the following were added: 1X SYBR Green (Takara Biotechnology Co., Ltd.) cDNA, 0.5 mM each primer and ROX (Takara Biotechnology Co., Ltd.). The conditions of the qPCR reaction were as follows: 50°C (2 min), 95°C (5 min), followed by 40 cycles of 95°C (15 sec) and 60°C (30 sec). The primers used were designed using Primer version 3.0 (26 (link)) and the sequences are listed in Table I. The relative expression levels of the target genes were calculated and normalized to the expression of GAPDH, a housekeeping gene.

Total RNA was extracted from isolated KCs using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. Reverse transcription of the purified RNA (2.5 μg) was performed using random primers and the AMV retrotranscriptase system (TakaRa, Dalian, Liaoning, China) according to the manufacturer’s protocol. SYBR Green real-time PCR was carried out using the ABI StepOne Plus (Applied Biosystems, Foster City, CA, United States). All reactions were performed in triplicate. In a final reaction volume of 20 μL, the followings were added: 1× SYBR Green (TakaRa), cDNA, 0.5 mmol/L of each primer, and ROX. The reaction conditions were as follows: 50 °C (2 min), 95 °C (5 min), followed by 40 cycles at 95 °C (15 s) and 60 °C (30 s). The primers used were designed with Primer 3.0 software and are listed in Table 1. The relative expression of target genes was calculated and normalized to the expression of the housekeeping gene GAPDH.