Phlebotomus perniciosus

Vieira, Cecilia Stahl, Bisogno, Sara, Salvemini, Marco, Telleria, Erich Loza & Volf, Petr, 2024, Azadirachtin disrupts ecdysone signaling and alters sand fly immunity, Parasites & Vectors (526) 17 (1), pp. 1-11 : 4

Phlebotomus perniciosus gene expression

Te expression of AMPs att, def 1, def 2, and the ecdysone-related genes EcR, Eip74EF, and Eip75B and serpent was evaluated through quantitative PCR. Quantitative PCR (qPCR) was prepared with 1 ng of cDNA samples, gene-specific primers, and SYBR Green PCR Master mix (Roche) in a 384-well plate to detect the expression of target genes using a LightCycler 480 thermocycler (Roche) in a final volume of 10 μl. Relative gene expression was calculated relative to P. perniciosus endogenous control genes GAPDH and GPDH [ 48] using the following cycling conditions: 95 °C for 10 min enzyme activation, initial denaturation at 95 ºC for 10 s, annealing at 60 °C for 20 s; extension at 72 °C for 45 s, repeated 45 times. Expression levels were expressed as the fold change compared to the control groups, using the Pfaffl method [ 52]. Te three independent experiments were performed with two pools of each sample in technical duplicates (n = 6).