The expressions of immune markers were evaluated through the relative quantification of transcripts induced by qRT-PCR using the comparative Ct (2−ΔΔCt) method, in which a relation was established
between the stimulation versus control conditions. In each situation,
the expression of the gene marker was normalized to the expression
of a HKG. Fig. 4A shows the effect on IL-1β transcript expression when
analyses were performed with the YWHAH and CD81 genes. It is interesting to note that at the level of transcript expression, IL-1β maintained
the tendency to increase over time, however, an approximately 40% decrease in expression was evidenced at 24 hpi as an effect of the HKG
employed (Fig. 4A). A very similar phenomenon was found for the immune markers IL-8, IL-15, IL18, INFβ, and IRF3 (Fig. 4B). Taken together,
these results demonstrated significant differences in transcript expression when using qRT-PCR as a method for quantifying transcripts, independent of the selected HKG (Fig. 4A and B). Finally, in the context of
BVDV-1 infection at 24 hpi, it was possible to observe a strong decrease
in IL-1β transcript levels, a result that would apparently be a consequence of an immunosuppression phenomenon triggered by viral infection (data not shown).