Total RNA was isolated from samples frozen in TRIzol Reagent® (Invitrogen) following the manufacturer’s instructions. A total of 1 µg of total RNA was treated with DNAse I (1 unit/µg RNA, Promega, Madison, WI, USA) to remove genomic DNA. The first strand of cDNA was synthesized by reverse transcription using the Superscript III (Invitrogen) with an oligodT12-18 primer, followed by RNAse H (Invitrogen) treatment for 60 min at 50 °C. The expression of the genes codifying for the AMPs, Hepcidin (hamp), complement factor 3-1 and 3-2 (c3), lysozyme (lyz), and Nk-lysin (nk-lys),was analyzed in real-time PCR, using an ABI PRISM 7500 instrument (Applied Biosystems) and SYBR Green PCR Core Reagents (Applied Biosystems), as previously described [36 (link)]. Reaction mixtures were incubated for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C, and finally, 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C. The specific primers were designed using the Oligo Perfect software tool (Invitrogen) and shown in Table 1. Before the experiments, the specificity of each primer pair was studied using positive and negative samples. A melting curve analysis of the amplified products validated the primers for specificity. Negative controls with no template were always included in the reactions. For each mRNA sample, gene expression was corrected by the geometric average of the endogenous elongation factor 1 alpha (ef1a) and ribosomal protein L13 alpha (l13a) coding gene content in each sample. This was, expressed as 2−ΔCt, where ΔCt is determined by subtracting the endogenous Ct geometric average value from the target Ct. The reference genes, ef1a and l13a, were chosen based on the stability of their Ct values and the stability of their pattern of expression.
In addition, a convectional PCR was performed using F2 and R3 primers (Table 1) [37 (link)], which are specific for the capsid NNV gene and able to detect the vaccine. Products were run in 1.5% agarose gel for visualization.
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