Spatial expression pattern of Rhopr-CRZR using quantitative PCR (qPCR)

Rhopr-CRZR transcript expression was examined in various tissues from 5th instar R. prolixus. Tissues were dissected from 4 to 6 weeks post-fed (as previous instar) insects in nuclease-free phosphate-buffered saline (PBS) (Sigma–Aldrich, Oakville, ON, Canada). To determine the expression of the Rhopr-CRZR transcript around ecdysis, the CNS was dissected from 4th instar R. prolixus 3 days before ecdysis (DBE), 2 DBE, 1 DBE, 2 h post-ecdysis (2 HPE) into 5th instar, 4 HPE, 1 day post-ecdysis (DPE), 3 DPE, and 6 DPE.

Total RNA was extracted from each tissue using PureLink® RNA Mini Kit (Life Technologies Corporation, Carlsbad, CA, USA) which was then used for cDNA synthesis with iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). The cDNA was diluted 10-fold and used as a template for the qPCR reaction. The Rhopr-CRZR gene and the reference genes (alpha-tubulin, beta-actin, and ribosomal protein 49) were amplified using primers designed over exon/exon boundaries (Supplementary Table 3; Paluzzi et al., 2010; Paluzzi and O'Donnell, 2012). The qPCR reactions were carried out on the CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada) using SsoFAST™ EvaGreen® Supermix (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). The amplification conditions were as follows: initial denaturation at 95°C for 30 s, 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 5 s, and extension at 72°C for 5 s. Three biological replicates with three technical replicates were performed including a no-template control. The melting curve analysis was performed and the qPCR products were run on a gel and cloned to confirm that the specific transcript was amplified. The relative expression levels were determined using the ΔΔCt method and the fold differences were normalized to the three reference genes using the geometric averaging of the transcript levels.