Sybr primer ex taq 2 kit

Total RNA was isolated and subjected to reverse transcription and qPCR analysis. β-actin served as internal control. The primer pairs used in this study are: 5′-atccaggctgtgctgtcc-3′ as forward primer and 5′-gaggatcttcatgaggtagtcg-3′ as reverse primer for β-actin; 5′-gaacgctccctctgcttgc-3′ as forward primer and 5′-aaactcaacctgaaaacgttaccttc-3′ as reverse primer for ORF7. The ΔΔCt method [16 ] for relative quantification of gene expression was used to determine viral RNA levels. For analyzing the inhibitory effect on RNA levels, SYBR Green real-time PCR was performed. Briefly, reverse transcription was carried out using Takara PrimerScript RT reagents kit in a volume of 10 μl at 37 °C for 15 min. One microliter of reverse transcription reaction mixture was used for qPCR by using gene-specific primers and master mix from SYBR Primer Ex Taq II kit (Takara). All reactions were performed in a 25 μl reaction volume. The reaction was carried out at 95 °C for 30 s, followed by 35 cycles at 95 °C for 30 s and 60 °C for 5 s. Relative amount of PRRSV RNA were normalized to β-actin mRNA. Amplification and detection of samples were performed with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

To verify the reliability and accuracy of the sequencing results, five DEGs (FABP3, CYP7A1, ANKRD22, SCD1, and PCK1) that were significantly enriched in the PPAR pathway were selected for qRT-PCR verification. Seven liver samples were selected from the HA and control groups. Liver tissue was used to isolate the RNA using TRIzol Reagent (Takara Bio Inc. Otsu, Shiga, Japan). The total RNA purity and concentration were measured using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA integrity was checked by 1.5% agarose gel electrophoresis. The A260/A280 ratio was expected to range from 1.8 to 2.0. cDNA synthesis was performed using a two-step method offered by the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Bio Inc. Otsu, Shiga, Japan). All specific primers for qRT-PCR (Supplementary Table S1) were designed using Primer 5.0 and synthesized by Sangon Biotech (Shanghai, China). The expression levels of the selected DEGs were measured using the SYBR Primer Ex Taq™ II Kit (Takara Bio Inc. Otsu, Shiga, Japan) in a LightCycler 96 instrument qRT-PCR system (Roche Molecular Biochemicals, Mannheim, BW, Germany). The results were normalized to the expression levels of chicken β-actin using the 2^−ΔΔCt method for quantification. All experiments were performed in triplicate.