Abi viia7 pcr machine

Total RNA from liver tissue was isolated using the Trizol method (Thermo Fisher) followed by DNase treatment. The RNA was reversely transcribed using the Takara high capacity cDNA synthesis kit. PCR primers were designed using Primer 5.0 software (Primer-E Ltd., Plymouth, UK) and quantitative PCR amplification was performed using an ABI-ViiA7 PCR machine (Applied Biosystems, USA). The amplification was programed to start at 95°C for 10 min, followed by 40 cycles of degeneration at 95°C for 30 s, annealing at 60°C for 34 s, and extension at 60°C for 1 min. Relative mRNA expression levels (fold change) of the target genes were analyzed by the 2^−△△Ct method based on q-PCR data of comparative critical threshold (Ct). The primers used in this study were listed below: PPARα, 5-GGGTACCACTACGGAGTTCACG-3 (sense) and 5- CAGACAGGCACTTGTGAAAACG-3 (anti-sense); CPT1α, 5- ACATCCCTAAGCAGTGCCAGTT-3 (sense) and 5- TCGTCCGGCACTTCTTGATC-3 (anti-sense); PGC1α, 5-CCCTGCCATTGTTAAGACC-3 (sense) and 5-TGCTGCTGTTCCTGTTTTC-3 (anti-sense); ACOX1, 5-GCCTGAGCTTCATGCCCTCA-3 (sense) and 5-ACCAGAGTTGGCCAGACTGC-3 (anti-sense); ACSL, 5-CTGGTTGCTGCCTGAGCTTG-3 (sense) and 5-TTGCCCCTTTCACACACACC-3 (anti-sense); GLUT4, 5-GGCTTTGTGGCCTTCTTTGA-3 (sense) and 5-CTGAAGAGCTCTGCCACAATGA-3 (anti-sense).

Total RNAs from adipose tissues or cells were isolated using TRIzol reagent (Life technology, 15596018) according to the manufacturer’s instructions. Single-stranded cDNA were synthesized with a reverse transcription kit (Thermo Fisher Scientific, K1622). mRNA levels were measured by qRT-PCR with SYBR-RT-PCR master mix (Roche, USA) with ABI ViiA7 PCR machine (Applied Biosystems, USA). GAPDH was used as a loading control. The analyses of the related gene expressions were performed with the relative quantification comparative CT method. The primer sequences for qRT-PCR were shown in Supplementary Tab. 1.

The changes in mRNA expression were evaluated by RT-qPCR in the liver tissue from BSA@Cu_2−xS NPs and the corresponding recovery groups. Total RNA was extracted from the livers of rats treated with 8 mg/kg LNPs and SNPs at the end of the dosing period (Day 14) and recovery period (Day 42) using a SteadyPure Universal RNA Extraction Kit (Accurate Biotechnology, Hunan, China) according to the supplier’s instructions. RNA was reverse transcribed into cDNA using an Evo M-MLV reverse transcription kit (Accurate Biotechnology, Changsha, China) carried out by a gradient PCR machine (Tprofessional Thermocycler, Biometra, Germany). The reaction program was set up as follows: 37 °C for 15 min, 85 °C for 5 s, and cool down to 4 °C. Real-time quantitative PCR (RT-qPCR) was performed using a SYBR Green Pro Taq HS premix kit (Accurate Biotechnology, Changsha, China) on an ABI ViiA7 PCR machine (Applied Biosystems, Life Technologies, USA). The thermocycler parameters were set up as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Ct values were used to analyze the difference between the dosing group and the control group. All primers used in this study are listed in Additional file 1: Table S1.