Transscript first strand complementary dna

ZO-1, Occludin and Claudin-1 mRNA expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted from the mouse colon using Trizol Reagent (Invitrogen Life Technologies, Grand Island, NY, catalog no. 15596026), and total RNA was reverse transcribed using TransScript First-Strand complementary DNA (cDNA) Synthesis SuperMix (TransGen Biotech, Beijing, China, catalog no. AT301), according to the manufacturer’s instructions. The PCR primers were designed according to the NCBI database (Table). PCR was performed in a StepOnePlus Real-Time PCR System (Oxoid, Thermo Fisher Scientific, MA, USA) using a SYBR Premix Ex Taq (Takara Bio, Japan, catalog no. RR42LR). Each sample was examined in triplicate, and normalized to β-actin with the 2^−ΔΔCT method [20 (link)].

Monocyte chemoattractant protein 1 (MCP-1), CCL-3, and CCL-5 mRNA expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted from splenic lymphocytes using Trizol Reagent (Invitrogen) and total RNA was reverse-transcribed using TransScript First-Strand complementary DNA (cDNA) Synthesis SuperMix (TransGen Biotech), according to the manufacturer’s instructions. The PCR primers were designed according to the NCBI database (Table 1) and PCR was performed with a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) using SYBR Green PCR Core Reagents (TransGen Biotech). Amplification of β-actin mRNA was used as an endogenous control. There were 10 replicates per group and each sample was assayed in triplicate and a mean value was calculated. Data were analyzed according to the 2^–ΔΔCt method and results were expressed as relative mRNA levels. Each group was assayed 18 times (three replicates per group and each sample assayed in sextuplicate).