Prime script rt reagent kit

For mRNA quantification, total RNA was extracted with ice-cold Trizol reagent (Sigma) and then reverse-transcribed to complementary DNA (cDNA) using Prime script RT reagent kit according to the manufacturer's protocol. Equal amounts of cDNA were used in real-time quantitative polymerase chain reaction (RT-qPCR). All the PCR reactions were carried out using SYBR Premix Ex TaqTM kit (Takara, Kyoto, Japan) and followed manufacturer's instructions. The 11β-HSD I and 11β-HSD II expression were measured by fluorescence quantitative RT-qPCR method. The β-actin gene was used as an internal control for normalization in parallel with each gene examined. The values obtained for the target gene expression were normalized to β-actin and quantified relative to the expression in control samples. The cDNA synthesis conditions were 37°C 15 min for reverse transcription reaction and 85°C 5 s for inactivation of the reverse transcription enzyme, then cooled to room temperature, and stored at −20°C. Amplification was performed with the Light Cycler 2.0 Real-Time Detection System (Roche, Hercules, CA, USA) using the following protocol: 40 cycles (5 s at 95°C and 34 s at 60°C) after an initial activation step for 30 s at 95°C. Primer sequences were shown in Table 1.

RNA was extracted with the Trizol reagent (Sigma). Then, the entire RNA of circNFATC3 and LDHA was reverse-transcribed to cDNA using the Prime Script RT reagent kit (Sigma). Meanwhile, miR-520h was reverse transcribed using the miRNA First-Strand Synthesis kit (Sigma). Next, cDNA was used for qRT-PCR with an SYBR Green kit (Sigma). GAPDH and RNU6 (U6) were used as endogenous controls. The primers were as follows: circNFATC3, F: 5′- ACCCTTTACCTGGAGCAAACC-3′ and R: 5′-TGTGGTAAGCAAAGTGGTGT-3′; NFATC3, F: 5′-TCCACCTCCATCTACTTTAACCA-3′ and R: 5′-TTGGGACCACCTAATGGGCT-3′; LDHA, F: 5′-GAGTGGAATGAATGTTGCTGGTGTC-3′ and R: 5′-CCAGGATGTGTAGCCTTTGAGTTTG-3′; miR-520h, F: 5′-TCGCGACAAAGTGCTTCCCT-3′ and R: 5′-GTGCAGGGTCCGAGGT-3′; GAPDH, F: 5′-TCCCATCACCATCTTCCAGG-3′ and R: 5′-GATGACCCTTTTGGCTCCC-3′; U6, F: 5′-CTCGCTTCGGCAGCACATATACT-3′ and R: 5′-ACGCTTCACGAATTTGCGTGTC-3′. Relative expression was processed using the 2^−△△Ct method.

Total RNA was extracted from LV tissue using TRIzol reagent and reverse transcribed into cDNA using a Prime Script RT Reagent Kit (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer's instructions. RT–qPCR was performed with a CFX96™ Real-Time System (Bio-Rad, Hercules, California, USA) using SYBR Green (SYBR Premix Ex Taq™ II; TaKaRa) for fluorescence quantification. In this study, the mRNA expression of tumor necrosis factor α (TNF-α), IL-1β, IL-6, monocyte chemotactic protein-1 (MCP-1), IL-4, IL-10, CD80, CD86, CD206, CD163, arginase-1 (Arg-1), resistin, and inducible nitric oxide synthase (iNOS) was measured. Relative mRNA expression levels were calculated using the 2^∆∆Ct method. The mean value of each transcript was normalized to GAPDH. The primers used in the experiments are listed in Table 1.