Real-Time PCR Analysis of Overexpressed Genes

The five overexpressed genes under chlorantraniliprole exposure were selected to determine their relative expression level by quantitative real-time PCR (qPCR) method at different developmental stages, within different tissues and dsRNA feeding of fall armyworm. Three replications were conducted for each sample. The designed primers for each specific genes were shown in Table 1. RNA was extracted as mentioned above, and 500-ng RNA with DNA-free was reverse-transcribed for synthesizing the first-strand cDNA using the PrimerScript RT Reagent Kit Perfect Real Time Kit (Takara, Dalian, China) based on the manufacturer’s instructions. The qPCR (20 µl) contained were performed in a mixture. The reaction was performed on an ABI 7500 Real Time PCR system (Applied Biosystems) with three biological replicates and two technical replications for each cDNA sample. GADPH was used as the reference gene for normalization of the expression levels (do Nascimento et al. 2015 (link)). The relative expression level for the selected target genes was calculated using the 2^−ΔΔCT method (Livak and Schmittgen 2001 (link)).