Color sybr green qpcr master mix

RAW cells were cultured following the protocol described in section 2.6.2. After three days of incubation, total RNA was extracted from the cells using the EZ-press RNA Purification Kit (EZBioscience, USA). The RNA (1 ​μg) was reverse-transcribed to cDNA using a Color Reverse Transcription Kit (EZBioscience, USA). qPCRs were run using 2 ​× ​Color SYBR Green qPCR Master Mix (EZBioscience, USA) per the manufacturer's protocol. The primers used in the experiment are listed in Table 2.Table 2

Primers used in qRT-PCR experiments (mouse origin).

Table 2

Gene	Forward primer sequence (5′–3′)	Reverse primer sequence (5′–3′)
Tumor necrosis factor alpha (Tnfa)	AATGGCCTCCCTCTCATCAGTT	CCACTTGGTGGTTTGCTACGA
Inducible nitric oxide synthase (iNos)	GCTTCTGGTCGATGTCATGAG	TCCACCAGGAGATGTTGAAC
Arginase 1 (Arg1)	AAGACAGCAGAGGAGGTG	AGTCAGTCCCTGGCTTAT
Interleukin-1 receptor antagonist (Il1ra)	CTCCAGCTGGAGGAAGTTAAC	CTGACTCAAAGCTGGTGGTG

EZ-press RNA Purification Kit (EZBioscience) was used to extract total RNA from cells. cDNA was acquired using Color Reverse Transcription Kit with gDNA remover (EZBioscience). Quantitative PCR reactions were conducted using 2 × Color SYBR Green qPCR Master Mix (EZBioscience). The sequences of the primers for quantitative reverse-transcription PCR (qRT-PCR) were shown in Supplementary Materials and Methods.

qPCR was performed according to standard protocols as previously described (19 (link)). Briefly, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA), and the concentration was measured using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). For messenger RNA (mRNA), RNA was reversely transcribed into complementary deoxyribonucleic acid (cDNA) using Color Reverse Transcription Kit (EZBioscience, USA), and qPCR was performed on Bio-Rad CFX-96 (Bio-Rad, USA) with Color SYBR Green qPCR Master Mix (EZBioscience, USA). The expressions of interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were normalized to GAPDH. The qPCR primers used in the study are listed in Table 1. For miRNA, the RNA was reversely transcribed into cDNA using Reverse Transcriptase M-MLV (RNase H-) (Takara, Japan). qPCR was performed on Applied Biosystems ViiA^TM7 Real-Time PCR System (ABI, USA) with AceQ qPCR SYBR Green Master Mix (Vazyme Biotech, Nanjing, China). The expressions of miR-302d-3p, miR-302b-3p, miR-302a-3p, miR-103-3p, 6_9856 (prediction), miR-199b-3p, miR-199a-3p, miR-92a-3p, miR-122-5p, and miR-744-5p were normalized to snoU6. The qPCR primers used in the study are listed in Table 2.