In an independent experiment, we selected the MAP (n = 4) and LAP (n = 4) from a group of 160 weaned pigs (Table S1 ). After dissection, the temporal lobe tissues were carefully excised and preserved in an ultra-low temperature refrigerator (Thermo Scientific, Waltham, MA, USA). Total RNA extraction was performed using the Trizol method (Vazyme, Nanjing, China). The integrity of the extracted RNA was assessed using 1% agarose gels (Vazyme, China), while RNA purity was determined via UV spectrophotometry (Thermo Scientific, USA).
Trizol method
Manufactured by Vazyme
Sourced in China
Trizol method is a widely used technique for the isolation and purification of total RNA from a variety of biological samples. It is a single-step liquid extraction method that utilizes a monophasic solution of phenol, chloroform, and guanidine isothiocyanate to effectively separate RNA from DNA and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Uv spectrophotometry, by Thermo Fisher Scientific (1 mentions)
Ultra low temperature refrigerator, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Hiscript 3 first strand cdna synthesis kit, by Vazyme (1 mentions)
Cfx96 real time pcr detection system thermocycler, by Bio-Rad (1 mentions)
Hiscript 2 one step qrt pcr sybr green kit, by Vazyme (1 mentions)
Sybr green master premix, by Vazyme (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
4 protocols using trizol method
1
Temporal Lobe RNA Extraction Protocol
2
Hc-HAP Gene Expression Profiling
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Detection of relative transcript levels of Hc-HAP genes in different life stages (adults, L3s, eggs and xL3s) was performed by quantitative real-time PCR (qPCR) using primers (Additional file 1 : Table S2) as previously described [12 (link), 21 (link)]. Briefly, total RNA was isolated from adults (female and male), eggs, L3s and xL3s by the TRIzol method (Vazyme Biotech Co., Ltd.). Then, 1 µg of RNA was synthesized into cDNA using the HiScript III First Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd.). The β-tubulin gene of H. contortus was used as a reference gene. The data were analyzed according to raw cycle thresholds (Ct), which were obtained using ABI Prism 7500 software (Applied Biosystems, Foster City, CA, USA) using the comparative Ct (2−ΔΔ Ct) method.
3
qRT-PCR Analysis of Intestinal Tissues
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First, the TRIzol method (Vazyme Biotech Co., Ltd., Nanjing, China) was used to extract total RNA from the intestinal tissues. Then, the quality and quantity of the RNA were checked with a NanoDrop 2000 spectrophotometer. Finally, the reaction system was set up according to the instructions of the HiScript® II One Step qRT-PCR SYBR Green Kit (Q221-01, Vazyme, Nanjing, China) and performed on a CFX96 real-time PCR detection system thermocycler (Bio-Rad). The specific primers for the reference gene (β-actin) and target genes in this experiment are displayed in Table 3 . The mRNA expression levels were calculated from the standard curve, normalized to β-actin, and quantified using the relative standard curve method.
4
Quantitative RT-PCR Analysis of THP-1 Cells
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Total RNA of THP-1 cells was extracted by Trizol method (Vazyme Biotechnology, Nanjing, China) and reverse transcribed into cDNA (Vazyme Biotechnology, China) for qRT-PCR detection (CFX96, Bio-Rad, Hercules, CA, USA). The total PCR reaction system included 10 μL SYBR Green Master premix (Vazyme Biotechnology, China), 0.4 μL (10 μmol/L) of upper and lower primers, and 2 μL cDNA template. The reaction process included initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 3 s, annealing at 58 °C for the 20 s, and extension at 72 °C for 30 s, with a total of 40 cycles. Using GAPDH as an internal reference, the relative gene expression was calculated by formula 2-ΔΔCt. The PCR primers’ sequences are shown in supplementary Table S1 .
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