qRT-PCR Analysis of Pathogens and Cytokines

The total RNA was isolated using TRIzol agent (TransGen Biotech, Beijing, China), and each RNA sample was reverse-transcribed to complementary DNA (cDNA) by the PrimeScript RT Reagent Kit (Takara, Dalian, Liaoning Province, China). cDNA was used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The sets of primer pairs of the two pathogens and of the nitric oxide synthase genes are listed in Table 1, and the primer pairs of cytokines can be found in Nang et al.’s paper [23 (link)]. For qRT-PCR reactions, the 25 μL reaction mixture included 2 μL cDNA, 12.5 μL SYBR Premix Ex TaqTM II (Takara, Beijing, China), 1.0 μL of forward primer and 1.0 μL of reverse primer, and 8.5 μL RNAase-free water (Takara, Beijing, China). The reaction conditions were 95 °C for 3 min, followed by 44 cycles of 95 °C for 10 s, then the specific melting temperature (Tm) of a primer pair for 30 s, and then 95 °C for 10 s and 72 °C for 10 s, using a Bio-Rad IQ5 Thermal Cycler (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as a reference gene. The expression fold changes were calculated using the 2^−△△Ct method [24 (link)].