Primescript rt reagent kit with gdna eraser

The total RNA of the jejunal tissues was extracted using the AG RNAex Pro reagent (Accurate Biology, Changsha, China). The purity and concentration of the extracted total RNA were determined using a NanoDrop ND-2000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, US). The total RNA (1 μg) was reversely transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Accurate Biology) for quantitative PCR analysis. The real-time quantitative PCR (RT-qPCR) was performed on the Light Cycler^® 480 II Real-Time PCR System (Roche, Basel, Switzerland). Primer sequences were synthesized by the Tsingke Biotechnology Co., Ltd. (Beijing, China) and used in this study (listed in Supplementary Table S1). The RT-qPCR was carried out in a 10-μL reaction system, consisting of 4.2 μl of cDNA template, 0.4 μl of each primer, and 5.0 μl SYBR^® Green mix (AG, Changsha, China). The RT-qPCR conditions were as follows: an initial step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 5 s and annealing at 60°C for 30 s, and a final extension at 72°C for 30 s. The relative mRNA expression levels were calculated using β-actin as the internal control and the 2^−ΔΔCt method (Rao et al., 2013 (link)).

The RNA was extracted from liver, ileum, jejunum, and spleen using TRIzol Reagent (0.1 g tissue per 1 mL TRIzol, Accurate Biology, Changsha, China). The purity and concentration of extracted total RNA were determined using a NanoDrop 2,000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). The total RNA (1 μg) was reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (Accurate Biology). Then 2.0 μL of cDNA template was mixed with the forward and reverse primers (0.25 μL, respectively), SYBR Green mix (5.0 μL), and RNase free water (2.5 μL). An RT-PCR analysis was performed on the Light Cycler^® 480 II Real-Time PCR System (Roche, Basel, Switzerland). The real-time PCR conditions were as follows: an initial step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and a final extension at 72°C for 30 s. Pig-specific primer sequences are shown in Supplementary Table 2 (Sangon Biotech, Shanghai, China). Target gene expression was calculated using the 2^−ΔΔCt value (19 (link)), and β-actin was used as the internal control.

The primer sequences were synthetized by RIBOBIO Corporation (Guangzhou, China). Trizol (Thermo Fisher Scientific Inc., MA, USA) method was employed to extract the total RNA in the cells in each group, and the concentration and purity of the RNA were measured. Samples were reacted in an Eppendorf PCR amplifier and the PCR amplification was conducted by SYBR^® Premix Ex Taq (Accurate Biotechnology Co., Ltd. Hunan, China) and a real-time quantitative PCR amplifier (Applied Biosystems QuantStudio 5, ABI Company, Oyster Bay, NY), according to the directions of PrimeScript RT reagent Kit with gDNA Eraser (Accurate Biotechnology Co., Ltd. Hunan, China). U6 (F: 5′-CGCTTCGGCAGCACATATAC-3′; R: 5′-TTCACGAATTTGCGTGTCAT-3′) was, respectively, used as the internal reference of miR-22-3p. The data were analyzed by 2^−ΔΔCT method.