Gene Expression Analysis by qPCR

Transcript levels of specific genes were measured by quantitative real-time PCR (qPCR). Total RNA was extracted from larvae using Ribozol reagent (VWR, Canada) according to the manufacturer’s instructions and quantified using a SpectraDrop Micro-Volume microplate (VersaMax, Molecular Devices, USA). One microgram of RNA was treated with DNase I (Thermo Scientific, USA) to remove genomic contamination prior to cDNA synthesis using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA), according to the manufacturer’s protocols. Transcript levels were measured by qPCR in duplicate using gene-specific primers as described previously^{23 (link)}. See^{21 (link)} for primer specific sequences and annealing temperatures.