Mosquito Dissection, RNA Extraction, and cDNA Synthesis

Dissections, RNA extraction, and cDNA synthesis were performed as described in Calkins et al. [11 (link)]. In brief, mosquitoes were anesthetized on ice before being dissected in mosquito Ringer solution (consisting of 150 mM NaCl, 3.4 mM KCl, 1.7 mM CaCl₂, 1.8 mM NaHCO₃, 1 mM MgCl₂, 5 mM Glucose, 25 mM HEPES; pH 7.1). Tissues were isolated, transferred to 1.5-ml micro-centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA), and preserved in TRIzol^® reagent at −80 °C until utilized in RNA isolation. Total RNA was isolated using the method of Chomczynski and Sacchi [17 (link)] and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). cDNA libraries were synthesized using 4 µg of total RNA and the GoScript^TM Reverse Transcriptase system with random primers (Promega, Madison, WI, USA), following manufacturer’s protocols. cDNA libraries were stored at −20 °C until needed for qPCR.