Sybr green real time pcr mix

Total RNA was isolated using Trizol method (Invitrogen). One μg of total RNA was reversely transcribed in a total volume of 10 μL with ReverTra Ace qPCR RT Master Mix kit (TOYOBO) following manufacturer's instructions. The cDNA was diluted three times, and 1 μL was used for real‐time PCR in a 20 μL reaction using SYBR Green Real Time PCR Mix (Qiagen). The PCR conditions were 95°C for 2 minutes, followed by 40 cycles of 95°C for 20’’ and 60°C for 10’’. All primers were listed in Table S1. The PCR efficiency for each primer pair was between 95% and 105%. The expression of target gene was normalized to that of GAPDH and calculated using 2^−∆∆Ct method as described in BioRad's qPCR manual.

Total RNA was isolated using TRIzol method (15596026, Invitrogen, USA). One microgram of total RNA was reversely transcribed in a total volume of 10 μl with ReverTra Ace qPCR RT Master Mix kit (FSQ-201, TOYOBO, Japan) following the manufacturer’s instructions. The cDNA was diluted 3 times, and 1 μl was used for real-time PCR in a 20-μl reaction using SYBR Green Real Time PCR Mix (204143, Qiagen, Germany). The PCR conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 20″ and 60 °C for 15″. All primers are listed in Table 1. The expression of target gene was normalized to that of GAPDH and calculated using the 2^-∆∆Ct method.

Quantitative real-time PCR (qPCR) was performed using SYBR Green real-time PCR mix (QuantiNova™, Qiagen™, Hilden, Germany) following the manufacturer’s instructions. For each sample, 3 µL of cDNA sample was added into 10 µL of 2X SYBR Green PCR master mix, 1.4 µL of each forward and reverse primers of immune-related genes (Table 3), and 1.2 µL of nuclease-free water into a PCR tube. Each cDNA sample group was prepared in triplicate. The samples were run in the Rotor-Gene Q real-time PCR machine (Qiagen™, Hilden, Germany). The resulting threshold cycle (CT) values were analyzed and recorded using Rotor-Gene Q software (Qiagen™, Hilden, Germany). The qPCR program was as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing/elongation at 60 °C for 60 s. A melting curve was run for each primer set to confirm the reaction specificity. The relative expression of each immune-relative gene was determined by comparing it with the expression level of the β-actin gene as the housekeeping gene using the Livak method, 2−ΔΔCT method.