Quantitative real time pcr system

Total RNA of S. japonicum was extracted using TRIzol Reagent (Thermo Fisher Scientific, Waltham, USA) as instructed by the manufacturer. Then, cDNA was synthesized from total RNA using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). The primers of 13 apoptosis-related genes are displayed in Table 1. NADH was used as the internal reference gene. In addition, total RNA of liver tissues was extracted. Liver fibrosis-related gene primers are shown in Table 2. GAPDH served as the internal reference gene. Real time quantitative PCR (RT-qPCR)was performed using SYBR® Premix Ex Taq ™ (Takara, Tokyo, Japan) in a 20- μL volume. The PCR was run on a real-time quantitative PCR system (Bio-Rad, California, USA) at 95°C for 30 s, followed by 95°C for 5 s and 60°C for 34 s for 40 cycles. Next, the melting curve was analyzed (95°C for 15 s and 65°C for 15 s.). The relative expression of each gene was calculated using the 2^−ΔΔCt method (18 (link)).

Total RNA was extracted from the ileum and colon using TRIzol Reagent (Invitrogen Life Technologies, Waltham, USA) according to the manufacturer's instructions. Subsequently, cDNA was synthesized from the total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Wilmington, USA). The qPCR primers were synthesized by Invitrogen Biotechnology (Carlsbad, CA, USA). GAPDH served as the internal reference gene. PCR was carried out on a real-time quantitative PCR system (Bio-Rad, California, USA) with an initial step at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 55 °C for 30 s. Subsequently, a melting curve was generated (65 °C for 5 s and 95 °C for 20 s). Relative gene expression levels were determined using the 2^−ΔΔCT method. Primer sequences were as follows: GAPDH: 5’-GGG TGT GAA CCA CGA GAA AT-3’ and 5’-CCT TCC ACA ATG CCA AAG TT-3’; Foxp3: 5’-CAC CTT TCC AGA GTT CTT CCA CA-3’ and 5’-CGG ATG AGG GTG GCA TAG GT-3’; IL-17: 5’-GGA CTC TCC ACC GCA ATG AA-3’ and 5’-TTT CCC TCC GCA TTG ACA CA-3’. Each PCR was performed in technical triplicates.

The RNA-seq data’s reliability was validated by means of qRT-PCR. RNA extraction and purification were undertaken, followed by cDNA synthesis using the RevertAid™ Kit (Fermentas). 16 genes were selected randomly for qRT-PCR analysis. To normalize the cDNA levels in each reaction, A. thaliana Actin gene was employed as endogenous control for relative expression calibration. Supplementary Table 1 contains the primer sequences utilized for the genes selected. Maxima SYBR Green Master Mix (Thermo Scientific) was used for qRT-PCR, which was conducted using the Real-time Quantitative PCR System (iQ5, Bio-Rad, USA), with three repetitions performed. The 2^−ΔΔCT method was utilized to analyze the relative expression data.