Total rna extraction kit

Total RNA from the tumor tissues, PSOs, and traditional organoids were extracted using the Total RNA Extraction Kit (#19221ES50, Yeasen Biotech, China), following the manufacturer’s protocol. qPCR was performed using the Accurate 96 Real-Time PCR machine (DLAB Scientific, Beijing, China) with the Hieff® qPCR SYBR Green Master kit (#11203ES08, Yeasen Biotech, China), as per the manufacturer’s instructions. All samples were set triplications (n≥3). The expression levels of the measured genes were normalized to the internal control and analyzed using the 2−ΔΔCt method. Primers of relevant genes have been listed in Table S1.

Total RNA was extracted using a Total RNA Extraction Kit (Yeasen Biotech, Shanghai, China) from the recombinant yeast. To obtain the first-strand cDNA using the 1st Strand cDNA Synthesis Kit (Yeasen Biotech, Shanghai, China). A 196-bp fragment of the VP2 gene was amplified by the forward primer (5′-CTACACTATAACTGCAGCCGAT-3′) and reverse primer (5′-CGCAGTCCCATCAAAGCCTA-3′) in a total volume of 20 μL with a TransStart^® Top Green qPCR SuperMix. To detect the expression patterns of immune-related genes, the RNA was extracted from the spleen and thymus of mice, respectively (n = 3 per group). The primers were listed in Table 1, and relative expression of the genes was normalized to that of β-actin. Data were analyzed based on the 2^−ΔΔCT method. The balance of Th2/Th1 and Th17/Treg at the level of master transcription factors was determined by measuring the expression of effector T-cell master transcription factors.

The primary cells and immortalized cell lines were verified for SCFA transport function. A total RNA extraction kit was used (Yeasen Biotechnology, Shanghai, China) to extract cellular total RNA. Gene expression was determined by qPCR as previously reported [40 (link), 41 (link)]. The Hifair™ II 1st Strand cDNA Synthesis Super Mix for qPCR kit was used (Yeasen Biotechnology, Shanghai, China) for reverse transcription. Reverse transcription was not less than 1 μg total RNA in a 20 μl reaction volume. The Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix kit was used (Yeasen Biotechnology, Shanghai, China) for qRT–PCR. qRT–PCR was performed using SYBR Green real-time PCR master mix in a qPCR system (QuantStudio 5, USA). RT–PCR cycles consisted of 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. β-Actin was used as the internal control. The PCR primers used in the quantitative assays are listed in Table 1. The fold change in mRNA expression was determined using the 2^−ΔΔCt method [42 (link)].

Total RNA was extracted from both in vivo and in vitro using the Total RNA Extraction kit (19211ES60, YEASEN, Shanghai, China). The reverse transcript (cDNA) was transcribed from total RNA according to the Fast King-RT Super Mix kit (Tiangen, Beijing, China). qRT-PCR was performed with the SYBR Green PCR Master Mix (11201E03, YEASEN, Shanghai, China). Results were quantified by the 2^−ΔΔCt method relative to the housekeeping gene actin. Primer pairs are listed in Supplementary Table S1.

Yak rumen fibroblasts were seeded in 6-well plates at a density of 1 × 10⁵ cells/mL, and challenged with LPS (Solarbio, Beijing, China) at different concentrations (0, 2, 4, 8, 16 and 32 μg/mL). Then, these cells were washed with PBS. Cellular total RNA was extracted using a total RNA extraction kit (Yeasen Biotechnology, Shanghai, China). To determine gene expression, qPCR was performed following previously reported methods [76 (link),77 (link)]. Reverse transcription was carried out using the SweScript All-in-One Blue RT SuperMix for qPCR (One-Step gDNA Remover) (Servicebio Biotechnology, Shanghai, China). A reverse transcription reaction volume of 20 μL was used, with 1 μg of total RNA. For qRT-PCR, the 2× Universal Blue SYBR Green qPCR Master Mix kit (Servicebio Biotechnology, Shanghai, China) was employed. The qPCR system (QuantStudio 5, Foster City, CA, USA) involved an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 30 s. The internal control for normalization was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the primer sequences used for the quantitative assays are provided in Table 1. The relative fold change in mRNA expression was determined using the 2^−ΔΔCt method [78 (link)].