2.5. Quantitative Polymerase Chain Reaction (qPCR) Validation of Differentially Expressed Genes

To validate expression profiles of DEGs determined from RNA sequencing analysis, qPCR was conducted on select genes involved in the top predicted pathways altered following bifenthrin treatment. RNA was diluted to 1 µg and reverse transcribed to cDNA using an iScript Reverse Transcription Supermix kit (Bio-Rad, Hercules, CA, USA). To perform qPCR, a 20 µL reaction was performed, with an input concentration of 100 ng cDNA and 10 µM of primer pairs (csf1, cbs, ptafr, and mtap; Supplementary Table S1). All qPCR reactions were conducted in triplicate and run on a Bio-Rad CFX Connect Real-Time PCR Detection System and followed the same thermal cycling conditions as previously described [23]. To determine expression fold change between treatment and control groups, 2^−ΔΔCt was used [36], with all results normalized to the housekeeping gene, ef1α, as no significant difference in expression was observed between treatments.