Superreal premix plus sybr green reagent kit

cDNA was synthesized from the total RNA using the FastQuant RT Kit as described above. The quantitative real time PCR (qPCR) analysis was carried out using SuperReal PreMix Plus (SYBR Green) reagent kit (TianGen, Beijing, China) (Huang et al., 2018 (link)). GhACT4 (accession number: AY305726) was used as an endogenous control (Supplementary Table 1). Three biological replicates were performed.

Total RNA was extracted from the colonic tissues from three group (n = 10) using TriQuick Reagent (Solarbio, Beijing, China), and quantified and purified using an ultra-micro UV visible spectrophotometer (NanoDrop 2000, Thermo, United States). The RNA was reverse-transcribed into cDNA using a PrimeScript^TM RT reagent kit with gDNA Eraser (TaKaRa, Japan) and stored at −80°C. The resulting cDNA was analyzed by conducting the real-time quantitative polymerase chain reaction with a SuperReal PreMix Plus (SYBR Green) reagent kit (TIANGEN, Beijing, China) with various primers (Supplementary Table S1). The relative expressions were determined using the 2-ΔΔCt method.

Take the liver tissue and grind it with liquid nitrogen, grind it into powder, transfer it into a 1.5 ml EP tube and mix with 1 ml Trizol, and centrifuge at 12,000 g for 10 min at 4°C. Add 120 ul of chloroform to the supernatant, centrifuge for 15 min at 4°C and 1,200 r, take 300 ul of supernatant into another centrifuge tube, add an equal volume of isopropanol to the supernatant, centrifuge at 4°C, 1,000 r for 10 min, remove the supernatant, add 600 ul of 75% ethanol to wash the precipitate, centrifuge at 8,000 r, 4°C for 10 min, remove the ethanol, add an appropriate amount of DNAase Free Water, and use a reverse transcription kit (gDNA Eraser) for reverse transcription. The resulting cDNA was analyzed by conducting the real-time quantitative polymerase chain reaction with a SuperReal PreMix Plus (SYBR Green) reagent kit (TIANGEN, Beijing, China) with various primers (Table 1).