Thermo nanodrop 2000 ultra trace visible spectrophotometer

Total RNA of snap-frozen liver was extracted using TRIZol reagent (TaKaRa, Otsu, Shiga, Japan, CAS: 9108). The RNA integrity was examined on 1% of agarose gel using GelRed staining. The RNA contents were quantified by Thermo NanoDrop 2000 Ultra Trace Visible Spectrophotometer (Thermo Fisher, Waltham, MA, United States). After that, 1,000 ng total RNA was reverse-transcribed into cDNA in a 20 μl reaction volume using the PrimerScript RT Reagent kit (TaKaRa, Otsu, Shiga, Japan, CAS: RR036A). Real-time PCR was performed on the QuantStudioTM Design & Analysis Software (Thermo Fisher, Waltham, MA, United States). Primers were synthesized by Invitrogen Biotech Co., Ltd. (Shanghai, China) and listed in Table 1. qRT-PCR was performed in a 20 μl reaction mixture using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China, CAS: Q311-02). The thermal profile was 3 min at 95°C, 10 s at 95°C for 40 cycles, and then 30 s at 60°C. The relative gene expression was calculated based on the 2^−ΔΔCT method after normalization to housekeeping gene GAPDH. Samples in the LFD group were used as calibrator.

Total RNA was isolated from snap-frozen liver tissues using TRIZol reagent (TaKaRa, Otsu, Shiga, Japan, CAS: 9108). The RNA concentration and absorbance at 260 and 280 nm, were quantified by Thermo NanoDrop 2000 Ultra Trace visible spectrophotometer (Thermo Fisher, Waltham, MA, USA). The RNA integrity was determined on 1% agarose gel with ethidium bromide staining. The mRNA was immediately reversed-transcribed into complementary DNA (cDNA) using the PrimerScript RT reagent kit (TaKaRa, Otsu, Shiga, Japan, CAS: RR036A) according to the manufacturer’s protocol. Real-time PCR was conducted in the ABI StepOnePlus^TM PCR system. The primer sequences are listed in Table 1 and synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). PCR reaction mixture of 20 μL was prepared using 0.4 μL each of forward and reverse primers, 0.4 μL of 50× ROX Reference Dye 2, 10 μL of 2× ChamQ SYBR qPCR Master Mix (Vazyme Biotechnology, Nanjing, China, CAS: Q311-02), 6.8 μL of double-distilled H₂O and 2 μL cDNA. The following thermal condition was used for qRT-PCR: 3 min at 95 °C, 40 cycles of 10 sec at 95 °C, and 30 sec at 60 °C. The relative mRNA expression was calculated by the 2^−ΔΔCt method after normalization with housekeeping genes GAPDH. Samples in the CON group were used as calibrators. The sequences of primers used in this experiment are shown in Table 1.

Total RNA was isolated from 50-mg liver samples using TRIZol reagent (TaKaRa, Otsu, Shiga, Japan, CAS: 9108) in accordance with the manufactures’ protocol. The concentration, integrity, and purity of extracted total RNA were quantified by Thermo NanoDrop 2000 Ultra Trace visible spectrophotometer (Thermo Fisher, Waltham, MA, USA). Subsequently, 1 µg of RNA were reverse-transcribed into complementary DNA (cDNA) using the PrimerScript RT reagent kit (TaKaRa, Otsu, Shiga, Japan, CAS: RR036A). With 2-μL diluted complement DNA, real-time quantitative PCR (RT-qPCR) were performed to calculate the expression of target genes utilizing the ABI StepOnePlus^TM PCR system. The RT-qPCR thermal profile was as follows: 3 min at 95 °C, 40 cycles of 10 sec at 95 °C, and 30 s at 60 °C. All primers that appeared in this study were showed in Table 1. The relative transcript level of target genes was assessed by the 2^−∆∆Ct method after selecting GAPDH as a reference gene.