Light cycler 96 real time pcr system

Sheep neutrophils and macrophages were seeded in six-well plates and cultured with F. necrophorum. The cells were collected at three different time points (2, 4 and 6 h), and total RNA was extracted using TRIzol (Ambion, TX, USA). The mRNA concentration was determined using a Titertek Berthold Colibri ultramicro spectrophotometer (Titertek-Berthold, Pforzheim, Germany). One microgram of mRNA was reverse transcribed into cDNA using a PrimeScript RT reagent Kit with a gDNA Eraser (TaKaRa, Beijing, China). RT-qPCR was performed with a TaKaRa TB Green Premix Ex Taq (Tli RNaseH Plus; TaKaRa, Beijing, China) on a light Cycler 96 Real-Time PCR System (Bio-Rad, Shanghai, China), β-Actin and GAPDH were used as housekeeping genes in neutrophils and macrophages, respectively. The reaction conditions were as follows: first, pre-denaturation at 95°C for 30 s, then denaturation at 95°C for 5 s, and annealing at 60°C for 30 s. The aforementioned steps involved 40 cycles at 95°C for 10 s. The dissolution curve was set as 65°C for 5 s, and 95°C for 5 s. The data were analyzed using the 2^-ΔΔCT method. The sequence of amplification primers is shown in Tables 1, 2. Each test was repeated three times.

For RT-qPCR, cDNA was synthesised from RNA using the iScript™ cDNA synthesis kit (Bio-Rad). RT-qPCR was performed on a LightCycler 96 Real-Time PCR system using SsoAdvanced™ Universal SYBR Green Supermix (Bio-Rad). Primer efficiencies were determined from cDNA dilution curves. The sequences, product sizes, optimal concentrations and efficiencies of primers used for RT-qPCR are listed in Table S3. Analysis of RT-qPCR data was carried out using the Pfaffl Method, which takes primer efficiency into account (Pfaffl, 2001 (link)).