Figure 4.

Validation of RNA-Seq using RT-qPCR. Eight representative genes (relish, lipoprotein receptor, dynamin, importin7, juvenile hormone epoxide hydroxylase 1; JHEH-1, DNAJ5, prophenoloxidase 1; PO1, and prophenoloxidase 2; PO2) were evaluated for their expression in hemocytes of shrimp under the NLHS and NH conditions in response to VP_AHPND infection and are referred to as NLHS-VP and NH-VP, respectively. Total RNA from hemocytes of NLHS-VP and NH-VP L. vannamei at 0, 6, and 24 hpi was used for cDNA synthesis. The relative expression levels of eight genes were determined by RT-qPCR and normalized against EF-1α, the internal reference. The relative expression ratio was calculated using the 2^−ΔΔCT method. The experiments were completed using triplicates. The expression level was calculated relative to that of the normal shrimp under the NH condition at 0 h after the VP_AHPND challenge. The bar graphs are the data from RT-qPCR presented as means ± standard deviations and the triangles (▲) are data from the RNA-Seq. Asterisks indicate significant difference (P < 0.05) from the respective VP_AHPND infected NH shrimp at 0 hpi.