4.5. qPCR and Statistical Analyses

qPCR was used to investigate the temporal and spatial expression analyses of Pf_C8α, Pf_C8β and Pf_C9 mRNA. The qPCRs of Pf_C8α, Pf_C8β and Pf_C9 were performed using a Roche LightCycler 480 (Roche, Mannheim, Germany). The gene-specific primer pairs for qPCRs were designed based on the amplified sequences of Pf_C8α, Pf_C8β and Pf_C9 genes (Table 2). The PCR reaction mixtures of Pf_C8α, Pf_C8β and Pf_C9 consisted of a 32-ng cDNA template, 10 μL LightCycler^® 480 SYBR Green I Master (Roche, Mannheim, Germany) and 1.0 μM of either gene-specific primer in a total volume of 20 μL. The qPCRs of Pf_C8α, Pf_C8β and Pf_C9 were performed in triplicate as: 95 °C for 5 min, followed by 45 cycles at 95 °C for 10 s, optimum annealing temperature (Table 3) for 10 s, 72 °C for 15 s respectively. At the end of each qPCR amplification reaction, melting curve analysis was performed to verify that each detected qPCR product was single. The amplification reaction without the cDNA template was used as a blank control.

Primers used for qPCRs of C8α, C8β and C9 mRNA and β-actin mRNA in Pelteobagrus fulvidraco.

T_a, annealing temperature; DL: real time PCR.

The relative expression levels of these three genes were calculated using the comparative C_t method with the formula 2^−ΔΔCt [58]. All data of qPCRs were expressed as the mean ± standard error (SE). One-way analysis of variance (one-way ANOVA) and Duncan’s post-hoc test were used to examine the differences among mean expression levels in different tissues and at early developmental stages using SPSS 17.0 software (IBM, Armonk, NY, USA). A t-test was performed to examine the differences in expression levels between the control and the experimental group after bacterial challenge. p-values less than 0.05 were determined to be statistically significant.