We further evaluated whether our RNA-Seq analysis results could be independently replicated using the quantitative reverse transcription polymerase chain reaction (RT-qPCR) technique as an independent tool. We randomly selected DE larval transcripts in our RNA-seq library and compared their relative fold changes to those we obtained for the same transcripts through RT-qPCR. Briefly, we generated five independent biological replicates (exposed and control) of the larvae, extracted and cleaned the respective total RNA libraries from our control and exposed larvae ab initio. We used the same methods and procedures we used to prepare the biological samples for the RNA-seq component of our study for sample preparations and total RNA extraction. We then reverse transcribed 1 μg of the total RNA using the iScript™ cDNA synthesis kit (BIO-RAD, Hercules, USA) on the Arktik thermal cycler (Thermo Scientific, USA), according to the manufacturer’s protocol.

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