Chamq universal sybr qrt pcr master mix

Ten DEGs involved in rhizome development were chosen for validation using quantitative real-time PCR (qRT-PCR). Primers for qRT-PCR were designed with Primer 3.0 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) (S1 Table). qRT-PCR was performed using the ABI StepOne^TM Plus Real-Time PCR System with ChamQ Universal SYBR qRT-PCR Master Mix (Vazyme Biotech, Dalian, China), and the products were amplified with a mixture of 1 μL of cDNA template, 2× ChamQ Universal SYBR qRT-PCR Master Mix, and 0.4 μL of each primer (10 μmol/μL) in water to a final volume of 20 μL. The amplification program consisted of one cycle at 95°C for 30 s, followed by 40 cycles at 95°C for 10 s and at 60°C for 30 s. Fluorescent products were detected in the last step of each cycle. A melting curve analysis was performed at the end of 40 cycles to ensure proper amplification of target fragments. The melting curve analysis consisted of one cycle at 95°C for 15 s and then at 60°C for 30 s, followed by one cycle at 95°C for 15 s. qRT-PCRs for each gene were performed for three biological replicates, with three technical repeats per experiment. Relative gene expression was normalized by comparison with the expression of Caucasian clover (c257504.graph_c0) and analysed using the 2^−ΔΔCT method [28 (link)].

FastPure Plant Total RNA Isolation Kit (RC401, Vazyme) was used to extract RNA, and then we used 1µg to synthesize cDNA (HiScript III 1st Strand cDNA Synthesis Kit, R312 Vazyme). ChamQ Universal SYBR qRT-PCR Master Mix (Q711, Vazyme) was used for qRT-PCR in ABI 7500 Fast Real-time PCR System (Applied Biosystems, USA). Gene-specific primers for qRT-PCR were designed by using primer-blast in NCBI, with melting temperatures of 55–60 °C, product lengths of 101–221 bp, primer length of 18–25 bp (Table S1). For qRT-PCR, the reaction contains 10 µL 2x ChamQ Universal SYBR qPCR Master Mix, 0.4 µL of each primer, 3 µL template, and ddH₂O to make up the total 20 µL volume. Then it was carried out in the following condition: one cycles of 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Each experiment was repeated three times, and two of the completed data were selected for drawing. Expression of all genes were calculated using a 2^−ΔΔCt method (Livak & Schmittgen, 2001 (link)).

MC-LR with a purity ≥ 95% was purchased from Alexis Corporation (Lausen, Switzerland). L. fermentum was acquired from Henan BeNa Culture collection (BNCC). Normal colonic epithelial NCM460 cells were purchased from Otwo Biotech Inc., (Shenzhen, China). RPMI 1640 medium was purchased from Sigma (Livonia, MI, USA). RIPA buffer, bovine serum albumin (BSA), and the Pierce bicinchoninic acid (BCA) protein assay kit were purchased from Thermo Scientific Pierce (Rockford, IL, USA). The polyvinylidene fluoride (PVDF) membrane was purchased from Merck Millipore Ltd., (Billerica, MA, USA). Trizol Reagent was purchased from TaKaRa LA Taq (Shiga, Japan). The ChamQ Universal SYBR qRT-PCR Master Mix and HiScript^® II Q RT SuperMix for qRT-PCR were purchased from Vazyme (Nanjing, China). The IL-6, TNF-α, IL-1β, and IL-10 ELISA kits were purchased from Neobioscience (Shenzhen, China). CSF1R antibody and Rap1b antibody were purchased from Abcam (Cambridge, UK). β-actin antibody and HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) were purchased from Proteintech (Wuhan, China).