Rnaiso plus reagent

NRF2, HO-1, and ACTB mRNA levels were measured using qRT-PCR. First, total RNA was extracted using the RNAiso Plus Reagent (Invitrogen, Carlsbad, CA, USA). Thereafter, reverse transcription was performed using the PrimeScript RT-PCR Kit (Takara Bio, Japan). qRT-PCR was performed on an Mx3000p real-time system (Stratagene, USA) using SYBR Premix Ex Taq (Yeasen, Shanghai, China). Amplification was initiated at 95°C for 30 s as the first step, followed by 40 successive cycles of 95°C for 5 s and 60°C for 20 s. Primers from Takara Bio are listed in Supplementary Table 1.

Total RNA from the cell was obtained using the RNAiso Plus Reagent (Invitrogen, Hong Kong, China) according to the standard guidelines. The Agilent 2100 Bioanalyzer system (Agilent Technologies, Carlsbad, CA, USA) was used for quality inspection, and only qualified RNA (A160/A180 = 1.6–1.8, concentration > 200 ng/uL) was used for the later trial. Reverse transcription of mRNA and miRNA was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China) and the Mir-X™ miRNA First-Strand Synthesis Kit (Takara) following the manufacturer’s protocol, respectively. Subsequently, qRT-PCR was performed in triplicate using the TB Green™ Premix Ex Taq™ II (Takara) on a CFX96 instrument (Bio-Rad, Hercules, CA, USA), and the relative expression levels of mRNA and miRNA were calculated using the 2^−ΔΔCt method. The mRQ 3′ primer in the Mir-X™ miRNA First-Strand Synthesis Kit (Takara) was used and served as a reverse primer for miRNA quantification, and U6 was used as an internal reference. Besides, GAPHD was used as an internal reference for mRNA quantification. Detailly, the primer sequences are listed in Table 2.

The total RNA from each sample was obtained using RNAiso Plus Reagent (Invitrogen, Carlsbad, CA, USA) according to the standard method. An Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used for the quality test and only eligible RNA (A160/A180 = 1.6~1.8, concentration > 200 ng/μL) was used for the later trial. A reverse transcription of the mRNA was performed using RT Easy^TM II (with gDNase) (FOREGENE, Chengdu, China). In addition, the RT-qPCR was performed in triplicate using Real-Time PCR Easy^TM SYBR Green I (FOREGENE, Chengdu, China) on a CFX96 instrument (Bio-Rad, Hercules, CA, USA). The relative levels of mRNA were calculated using the 2^−ΔΔCt method. β-Actin was used as the internal reference for the mRNA quantification. The sequence information is listed in Table 2.

Total RNA from the liver sample was extracted using RNAiso Plus Reagent (Invitrogen, Hong Kong, China), following the guidelines of the manufacturer. Subsequently, the purity, concentration, and integrity of RNA were determined by Agilent 2100 Bio-analyzer system (Agilent Technologies, Carlsbad, CA, United States), and only RNA meeting quality criteria (A260/A280 = 1.6–1.8; concentration ≥200 ng/μl) was used for the trial. Reverse transcription of mRNA and miRNA was performed using the RT EasyTM II (With gDNase) (FOREGENE, Chengdu, China) and the Mir-XTM miRNA First-Strand Synthesis Kit (Takara, Dalian, China), respectively. Then, qRT-PCR was performed in triplicate using the 2*TSINGKE Master qPCR Mix (SYBR Green I) (Tsingke, Chengdu, China) on a CFX96 instrument (Bio-Rad, Hercules, CA, United States), and the relative levels of mRNA and miRNA were calculated using the 2^−ΔΔCt method. The mRQ 3′ primer in the Mir-XTM miRNA First-Strand Synthesis Kit (Takara, Dalian, China) was served as a reverse primer for miRNA quantification, and U6 was used as an internal reference. Besides, GAPHD was used as the internal reference for mRNA quantification. The sequences of primers were showed in Table 1 and synthesized by Gene Pharma (Shanghai, China).

Total RNA from perirenal adipose tissue samples was extracted using RNAiso Plus Reagent (Invitrogen, Hong Kong, China), following the guidelines of the manufacturer. Subsequently, the purity, concentration, and integrity of RNA were determined by Agilent 2100 Bioanalyzer system (Agilent Technologies, Carlsbad, CA, U.S.A.) and Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, U.S.A.), and only RNA meeting quality criteria (A₂₆₀/A₂₈₀ = 1.6–1.8; concentration ≥ 200 ng/µl; RNA integrity number > 7) was used for the trial. Reverse transcription of miRNAs was performed using the Mir-X™ miRNA First-Strand Synthesis Kit (TakaRa, Dalian, China), following the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) was performed in triplicate using the TB Green™ Premix Ex Taq™ II (TakaRa, Dalian, China) on a CFX96 instrument (Bio-Rad, U.S.A.), and relative expression levels of miRNAs were calculated by 2^−ΔΔC_T method.