Sybr green premix pro taq hs qpcr kit

Total RNAs were isolated from KCs using TRIzol reagents (Invitrogen, USA). Complementary DNA synthesis was performed using 1 µg total RNA via HiScriptII 1st Strand cDNA Synthesis Kit or miRNA 1st Strand cDNA Synthesis Kit (Vazyme, China). qRT-PCR was carried out using SYBR Green Premix Pro Taq HS qPCR Kit on ABI 7500 Thermal Cycler (Thermo, USA) with the following steps: 95 ℃ for 5 min and 40 cycles of 95 ℃ for 10 s, 60 ℃ for 30 s. The relative expression level was normalized to U6 small nuclear RNA (snRNA) or GAPDH using the 2^−ΔΔCt formula. The details of primers used for qRT-PCR are summarized in Table 1. Statistical analysis data were taken from three independent experiments.Table 1

Primers for qRT-PCR

Primer	Sequence(5ʹ-3ʹ)
miR-374b-5p	GCGCGATATAATACAACCTGC
Universal reverse primer	GCTGTCAACGATACGCTACG
C/EBP β forward primer	GCTGAGCGACGAGTACAAGATGC
C/EBP-β reverse primer	CTTGTGCTGCGTCTCCAGGTTG
IL6 forward primer	CTTCTTGGGACTGATGCTGGTGAC
IL6 reverse primer	TCTGTTGGGAGTGGTATCCTCTGTG
iNOS forward primer	CCTAGTCAACTGCAAGAGAA
iNOS reverse primer	TTTCAGGTCACTTTGGTAGG
TNFα forward primer	CGCTCTTCTGTCTACTGAACTTCGG
TNFα reverse primer	GTGGTTTGTGAGTGTGAGGGTCTG
Arg1 forward primer	AGACAGCAGAGGAGGTGAAGAGTAC
Arg1 reverse primer	AAGGTAGTCAGTCCCTGGCTTATGG
GAPDH forward primer	CCACTCACGGCAAATTCAAC
GAPDH reverse primer	CTCCACGACATACTCAGCAC

The expression of Claudin-1, ZO-1, TNF-α, IL-22, IL-6, IL-1β, CAT, Nrf2, HO-1, and NQO1 in duodenal tissues was assessed using the real-time quantitative (qPCR) method. Duodenal tissues were mixed with RNAiso Plus, homogenized, and total RNA was extracted. The primer sequences are shown in Supplementary Table S1. The removal of genomic DNA was performed using 5× gDNA Clean Buffer and RNase-free water. Subsequently, reverse transcription was carried out using Evo M-MLV RT Premix according to the manufacturer’s instructions for qPCR. Subsequently, reverse transcription was carried out using Evo M-MLV Mix kit with gDNA Clean for qPCR Premix according to the instructions. The SYBR@ Green Premix Pro Taq HS qPCR Kit AG11701 was mixed with cDNA, and the reaction was carried out using the Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with 1 cycle of the pre-denaturation reaction: 95 °C for 30 s and 40 cycles of the polymerase chain reaction. The mRNA expression levels of target genes were calculated using the 2^−ΔΔCT method. Each group detected 6 values for each indicator, and the final average was taken as the indicator detection result.