Transscript one step gdna removal and cdna synthesis supermix reagent kit

The mRNA expression of inflammation-related genes (TLR4, MyD88, NF-κB, IL-6, IL-8, IL-10, and TNF-α) in the TLR/MyD88/NF-κB signaling pathway in the colon was determined by quantitative real-time PCR (qRT-PCR). The total RNA was isolated using the TransZol Up Reagent (TransGen Biotech, Beijing, China). In brief, cDNA was synthesized using a TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix reagent kit (TransGen Biotech, Beijing, China) according to the kit's instructions. Then, it was stored at −20°C for SYBR Green qRT-PCR. Gene-specific primers of all genes were designed using the Primer Premier software (PREMIER Biosoft International, CA, USA). The housekeeping gene GAPDH was used as an internal reference, and the primer sequences are shown in Table 2. The RT-qPCR profiles were as follows: 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, 60°C for 60 seconds, and extension at 95°C for 15 seconds.

The mRNA transcription levels of TLR4/MyD88/NF-κB signal pathway-related genes (TLR4, MyD88, NF-κB, IL-6, IL-8, and TNF-α) in the thymus were determined by qRT-PCR assay according to the manufacturer's instructions. Briefly, total RNA was isolated using the TransZol Up Reagent (TransGen Biotech, Beijing, China). Then, cDNA was synthesized using a TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix reagent kit (TransGen Biotech, Beijing, China) according to the kit's instructions and stored at −20°C for SYBR Green qRT-PCR. Gene-specific primers of all the genes were designed using the Primer Premier software (PREMIER Biosoft International, CA, United States). The GAPDH gene was used as an internal reference, and the primer sequences are shown in Table 3. The qRT-PCR profiles were as follows: 95°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for 60 s, and extension at 95°C for 15 s. All the reactions were carried out using an ABI 7900HT machine (Applied Biosystems, United States).

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the mRNA expression of inflammation and antioxidant-related genes. Briefly, the total RNA was isolated using the TransZol Reagent (TransGen Biotech, Beijing, China). The complementary DNA (cDNA) was synthesized using a TransScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix reagent kit (TransGen Biotech, Beijing, China) according to the kit's instructions. Then, it was stored at −20°C for SYBR Green qRT-PCR. All primer sequences were designed by OLIGO7 and presented in Supplementary Table 2. Relative gene expression levels were assessed using the 2^−ΔΔCT method and normalized to the housekeeping genes GAPDH and β-actin.