qRT-PCR Analysis of O. furnacalis Genes

For qRT-PCR analyses, total RNA was extracted from O. furnacalis larvae and adults using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Ambion, Austin, TX, USA), according to the manufacturer’s instructions. cDNAs were synthesized using the Omniscript Reverse Transcriptase kit (Qiagen, Hilden, Germany) in a 20 μL reaction mixture containing 1 μg total RNA. qRT-PCR analysis for OfMasc and Ofdsx mutants was performed using a SYBR Green Real-Time PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on an Eppendorf Real-Time PCR System. The PCR conditions were as follows: initial incubation at 95 °C for 5 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min. O. furnacalis actin was used as an internal control [32 (link)]. The gene-specific primers used for qRT-PCR are listed in Table 2.