Tripure reagent kit

Total RNA of eWAT or adipocytes were extracted with TRIpure Reagent kit (Takara, China) according to previous study (41 (link)). 500 ng of total RNA was reverse transcribed using M-MLV reverse transcriptase kit (Invitrogen, USA, 28025013). Primers were synthesized by Invitrogen (China). Quantitative PCR was performed in 25 µL reaction system containing specific primers and AceQ qPCR SYBR Green Master Mix (Vazyme, China, Q111-02). Amplification was performed in the ABI StepOne Plus™ RT-PCR System (CA, USA). The levels of mRNA were normalized in relevance to GAPDH. The expression of genes was analyzed by method of 2^−ΔΔCt.

Total RNA from adipose tissues and cells were extracted with TRIpure Reagent kit (Takara, China). 400 ng of total RNA was reverse transcribed using M-MLV reverse transcriptase kit (Takara, China). Primers were synthesized by Shanghai Sangon Ltd (Shanghai, China). Quantitative PCR was performed in 25 μL reaction system containing specific primers and SYBR Premix EX Taq (Takara, Dalian, China). The levels of mRNA were normalized using β-actin. The expression of genes was analyzed by method of 2^−ΔΔCt.

Total RNA was extracted from eWAT or adipocytes with TRIpure Reagent kit (Takara, Dalian, China). Reverse transcription of 500 ng total RNA using M-MLV reverse transcription kit (Takara, Dalian, China). Moreover, primers were synthesized by Invitrogen (Invitrogen, Shanghai, China). Further quantitative PCR was performed in 25-μL reaction system containing specific primers and AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). Amplification was performed in the ABI Step One Plus™ RT-PCR System (ABI, Carlsbad, CA, USA). The levels of mRNA were normalized in relevance to GAPDH. Moreover, the expression of genes was analyzed by method of 2^−ΔΔCt, ΔΔ = (CT_{Target Gene} − CT_GAPDH) T − (CT_{Target Gene} − CT_GAPDH) C.

Total RNA was isolated using TRIpure Reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. cDNA was generated by using the High Capacity cDNA Reverse Transcription Kit (Takara, Dalian, China). The process for qRT-PCR was performed as described previously [60 (link)].

Total RNA was extracted with TRIpure Reagent kit (Takara, China). 500 ng of total RNA was reverse transcribed using M-MLV reverse transcriptase kit (Takara, China). Primers were designed as Table 1 in Supplementary Data synthesized by Invitrogen (Shanghai, China). Quantitative PCR was performed in 20 μL reaction system containing specific primers, cDNA and SYBR Premix EX Taq (Takara, China). The levels of mRNA were normalized using β-actin. The expressions of genes were analyzed by the method of 2^−ΔΔCt.

Total RNA was extracted from adipose tissues (iWAT and iBAT) or white adipocytes using TRIpure Reagent kit (Takara, Dalian, China) according to the manufacturer's instructions. 500 ng of total RNA was reverse transcribed using M-MLV reverse transcriptase kit (Takara, China). Primers were designed by Premier 5.0 and were synthesized by Invitrogen (Table 1, Shanghai, China). Real-time PCR amplification was performed in 25 μL reaction system containing specific primers and SYBR Premix (Vazyme, Nanjing, China) under specific amplification conditions. Amplification reactions were carried out in the ABI StepOne plus™ RT-PCR System (Carlsbad, CA). The levels of mRNA were normalized to Gapdh. 2^−ΔΔCt method was used to analyze the data.