Chamq universal sybr pcr master mix

Total RNAs were extracted from adipose tissues using Trizol reagent (Invitrogen, USA) and reverse-transcribed into cDNA using reverse transcription Kit (R323-01, vazyme, China) according to the manufacturer's instructions. RT-PCR was performed with a ChamQ Universal SYBR PCR Master Mix (Q711-02, vazyme, China) by ABI QuantStudio™ 6 Flex system (Thermo Fisher Scientific, MASS, USA). The specific primers are listed in Table 1. The mRNA expression was relatively quantified according to CT value and calculated by 2^−∆∆Ct method.Table 1

Primers used for quantitative real-time PCR analysis

Primer name	Species	Sequence forward (5'to3')	Sequence reverse (5'to3')
IL10	Mouse	GCTGAGGCGCTGTCATCGATTT	GGCCCTGCAGCTCTCAAGTGT
IL6	Mouse	TCAATTCCAGAAACCGCTATGA	CACCAGCATCAGTCCCAAGA
IL1β	Mouse	CGTGCTGTCGGACCCATATGAG	GCCCAAGGCCACAGGTATTT
IL4	Mouse	TCACTGACGGCACAGAGCTA	CTGTGGTGTTCTTCGTTGCTG
TNFα	Mouse	ACGTCGTAGCAAACCACCAA	ACCCTGAGCCATAATCCCCT
PPARγ	Mouse	TCGCTGATGCACTGCCTATG	GAGAGGTCCACAGAGCTGATT
C/EBPα	Mouse	CAAGAACAGCAACGAGTACCG	GTCACTGGTCAACTCCAGCAC
SREBP-1c	Mouse	GAGCGAGCGTTGAACTGTAT	ATGCTGGAGCTGACAGAGAA
Adiponectin	Mouse	TGTTCCTCTTAATCCTGCCCA	CCAACCTGCACAAGTTCCCTT
GAPDH	Mouse	ACAACTTTGGCATTGTGGAAG	GTTGAAGTCGCAGGAGACAAC

Total RNA was extracted using the Total RNA Extraction Kit (Solarbio, Beijing, China). Subsequently, 1000 ng of the total RNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix Kit (Vazyme, Jiangsu, China). Quantitative real-time PCR was then conducted using the ChamQ Universal SYBR PCR Master Mix (Vazyme, Nanjing, China) and a StepOne Plus PCR system (Thermo Fisher, Massachusetts, USA). The quantitative real-time PCR employed the following primer pairs: Jagged1, 5′-GCCTCCTGTCGGGATTTGG-3′(forward) and 5′-AGTGATCGCCTGCATAGCCA-3′(reverse); Hes1, 5′-AAAAATTCCTCGTCCCCGGT-3′(forward) and 5′-GAATGCCGCGAGCTATCTTTCT-3′(reverse); Hey1, 5′-CGGACGAGAATGGAAACTTGA-3′(forward) and 5′-CTTGCTCCATTACCTGCTTCTC-3′(reverse); Nanog, 5′-CAATGGTGTGACGCAGGGATG-3′(forward) and 5′-CTGGCAGGAGAATTTGGCTGG-3′(reverse); OCT4A, 5′-ACCGAGTGAGAGGCAACCTG-3′(forward) and 5′-ACACTCGGACCACATCCTTCT-3′(reverse); ALDH1A1, 5′-ATGCTCACCCCACCTTCTTCA-3′(forward) and 5′-CAGTGGTAAGGTTTCTCACCTGT-3′(reverse); SOX2, 5′-GCGGAAAACCAAGACGCTCA-3′(forward) and 5′-CCGTTCATGTGCGCGTAACT-3′(reverse); GAPDH, 5′-ATGAATGGGCAGCCGTTAGG-3′(forward) and 5′-CCCAATACGACCAAATCAGAGAA-3′(reverse); GAPDH was used as an internal control.