Quantitative PCR Analysis of Porcine Viral Antigen-Stimulated Splenocytes

Splenocytes were placed into a 24-well plate at 1 × 10⁷ cells/well and then restimulated with inactivated PrV antigen (5 × 10⁵ TCID₅₀) at 37°C in 5% CO₂ atmosphere for 15 h. Cells were collected after centrifugation (1500 rpm, 4°C, 5 min) and washed with ice-cold PBS. EASY spin Total RNA Extraction Kit (TaKaRa, Dalinan, China) was used to isolate total RNA. Then, PrimeScript™ RT reagent kit (TaKaRa, Dalinan, China) was used to convert total RNA into cDNA. The primers, synthesized by Sangon Biotech, met the NCBI/Primer-BLAST standards and their sequences are listed in Table 1. Quantitative PCR was performed using SYBR Premix Ex TaqTM II (Tli RNaseH Plus) on ABI7300 (PE Applied Biosystems, USA) and data were determined using comparative C_T method (2^-△△C_T) [33 (link)–35 (link)], where C_T means the fractional cycle number at which the amount of amplified target reaches a fixed threshold, and formula −△△C_T = −(△C_T·Target − △C_T·Control), which the relative levels of the target gene mean the fold change of target gene expressions related to the untreated control [36 (link)].