Total RNA was extracted from all samples using the sex-biased sampling as described in Vea et al. (2016) (link). Each sample consisted of 0.5 to 2 mg of pooled individuals homogenized in TRIzol reagent, total RNA was extracted using nuclease-free glycogen (Thermo Fisher Scientific) as a carrier, and reverse transcribed using the PrimeScript RT reagent Kit with the gDNA Eraser (Takara Bio).
Expression profiles for the post-oviposition development of males and females were established by quantifying the levels of transcripts for targeted fragments using absolute quantitative RT-PCR (qRT-PCR), performed on a Thermal Cycler Dice Real Time System (model TP800, Takara Bio) as described previously (Vea et al., 2016) (link). Six serial dilutions of a plasmid containing a fragment of each gene was used as the standard. Primer sequences for each PkE93 isoform used for qRT-PCR are listed in Table S1. The values obtained by the second derivative maximum (SDM) method were normalised with the ribosomal protein L32 (rpL32) transcript levels. Primers for PkrpL32, our reference gene, were from our previous study (Vea et al., 2016) (link).
Expression profiles for the post-oviposition development of males and females were established by quantifying the levels of transcripts for targeted fragments using absolute quantitative RT-PCR (qRT-PCR), performed on a Thermal Cycler Dice Real Time System (model TP800, Takara Bio) as described previously (Vea et al., 2016) (link). Six serial dilutions of a plasmid containing a fragment of each gene was used as the standard. Primer sequences for each PkE93 isoform used for qRT-PCR are listed in Table S1. The values obtained by the second derivative maximum (SDM) method were normalised with the ribosomal protein L32 (rpL32) transcript levels. Primers for PkrpL32, our reference gene, were from our previous study (Vea et al., 2016) (link).