Chamq sybr color qpcr master mix

Total RNA was extracted from mycelia of S. eturmiunum growing in PDB (Potato Dextrose Broth) cultures using the Fungal RNA Kit (OMEGA Biotechnology, USA). cDNA was generated using the HiScript II QRT SuperMix for qPCR (Vazyme, Nanjing, China). The qRT-PCR was carried out using the 2 × ChamQ SYBR Color qPCR Master Mix (Vazyme, China) and performed on an ABI QuantStudio^TM 6 Quantitative Real-Time PCR System (Applied Biosystems). The specific primers of qRT-PCR listed in the Table 1. Changes in the relative expression level of each gene were calculated by the 2^−∆∆CT method [35 (link)]. Gene expression levels were normalized using the housekeeping gene actin. This experiment was repeated at least three times.

The cDNA obtained by reverse transcription was carried out qRT-PCR as a pre-experiment. According to the Ct values obtained in the pre-experiment, 2 μL cDNA was then taken, and the Ct value of cDNA was diluted to 20 μL with dd water as the optimal cDNA concentration required for the qRT-PCR reaction of different target genes. The software Primer Premier 5.0 was used to design qRT-PCR primers. The primers used in the experiment were listed in Table S2. The qRT-PCR reaction mixture was mixed using a ChamQ SYBR Color qPCR Master Mix Kit (Vazyme, Q431-02, China), which was 20 μL including 10 μL 2× ChamQ SYBR Color qPCR Master Mix, 0.4 μL forward and reverse primer of the target gene, respectively, 8.2 μL DEPC-treated water and 1 μL cDNA. The pre-denaturation temperature of PCR amplification was 95 °C for 5 min, followed by 40 cycles (95 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s), and then the determination of the melting curve (95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s). The β-actin gene was used as a reference gene for the qRT-PCR.

Using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) total RNAs of the liver and brown fat were isolated (n = 6 group; samples from mice were randomly selected). cDNA was obtained by reverse transcription of total RNAs with HiScript Reverse Transcription kit (Vazyme, Jiangsu, China). The qPCR was conducted with the 2×ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) on a LightCycler 480 system (Roche Applied Science, Indianapolis, IA, USA) with specific mouse primers. Results were normalized to the housekeeping YWHAZ gene and calculated based on the 2^−∆∆Ct method. All the primer sequences are shown in Supplemental Table S1.

Total RNA from colonic samples was extracted using the FastPure Cell/Tissue total RNA isolation kit (Vazyme Biotech Co., Ltd.). The cDNA generated by HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd.) was analyzed using primers for the indicated genes. Real-time quantitative PCR (RT-qPCR) was performed with 2×ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech Co., Ltd.) and run in a LightCycler 480 system (Roche) according to the manufacturer’s instructions. Genes Foxp3, Il6, Tnfa, Il23p19, Il10, Il17a, Il22, Il1b, Tgfb1, Rorgt, Zo1, Occludin, and Jam-1 were evaluated, and β-actin was used as the housekeeping gene for colonic tissue. Relative expression of mRNA levels was quantified using the threshold cycle (2^−ΔΔCT) method as described previously (54 (link)). The primers are shown in Table 1.

Total RNA was extracted from various plant organs of the WT using RNAprep Pure Plant Kit (TIANGEN) and was reverse transcribed using HiScript II Q RT SuperMix (Vazyme). Quantitative real-time PCR analysis was performed on an Applied Biosystems 7500 Real-Time PCR System with 2 × ChamQ SYBR Color qPCR Master Mix (Vazyme) and the data were calculated using the 2^−∆∆Ct quantification method. The rice Ubiquitin gene was used as an internal control to normalize gene expression. All primers used are listed in Additional file 1: Table S1.

The total RNA from the colon tissues was extracted by using TRIzol (azyme Biotech Co., Ltd.). Total RNA was purified by lithium chloride precipitation (51 (link)). RT-PCR was performed with 2×ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech Co., Ltd.) according to the manufacturer’s instructions. Relative mRNA expressions were quantified using the threshold cycle (2^−ΔΔCT) method as described previously (49 (link)). The primers are shown in Table S1.

Total colonic RNA was extracted by using TRIzol (Vazyme Biotech Co., Ltd) and subsequently purified through lithium chloride precipitation [19 (link)]. Real-time quantitative PCR (RT-PCR) was performed with 2 × ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech Co., Ltd.) following the manufacturer’s protocols. β-actin was used as the housekeeping gene for colonic tissue. The quantification of mRNA levels was measured using the threshold cycle (2^−ΔΔCT) method as described previously [20 (link)]. The primers are shown in Supplementary Table 1.