Primerscript rt reagent kits with gdna eraser

Male and female adults of 1, 5, 10, and 13 days (mated and unmated) after copulation were anesthetized with CO₂ and different tissues such as antennae, wings, legs, abdomens, and heads were separated. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol, and the RNA concentration was determined with an ultraviolet spectrophotometer (Eppendorf BioPhotometer Plus, Germany), before reverse transcription. cDNA for qPCR was synthesized using PrimerScript RT Reagent kits with gDNA Eraser (Takara Bio, Otsu, Japan).

Total RNA was extracted from individual samples by using the TRIzol reagent (Invitrogen, CA). Then quality and quantities were examined by using 1.0% agarose gel electrophoresis and ultraviolet spectrophotometer (Eppendorf Bio Photometer Plus, Germany). The first-strand cDNA for RT-PCR and qRT-PCR were synthesized from 1 μg of total RNA using MBI RecertAid First Strand cDNA kit (MBI Fermentas, Glen Burnie, MD, USA) and PrimerScript RT Reagent kits with gDNA Eraser (Perfect Real Time; Takara) respectively, according to manufacturer's instructions. The synthesized cDNA was stored at −20°C for future use.

To analyze the abundance of MsepCSP8 transcripts, total RNA was extracted from larvae (6th instar), pupae (5 days old), the whole bodies (3 days old), and body tissues (antennae, head, thorax, abdomen, legs and wings) of both sexes (1–5-days-old) using TRIzol reagent (Invitrogen, Carlsbad, CA, United States). To investigate the efficacy of RNAi on MsepCSP8 expression levels, total RNA was extracted from whole bodies at 1, 2, 3, 4, and 5 days post-eclosion from non-injected controls, dsGFP- and dsRNA-injected male and female adults. The purity of RNA was quantified by agarose gel electrophoresis and the concentration was determined using a spectrophotometer (Nanodrop-2000, Massachusetts, United States). The first-strand cDNA for RT-PCR and RT-qPCR were synthesized from 1 μg of total RNA using MBI RevertAid First Strand cDNA kit (MBI Fermentas, Glen Burnie, MD, United States) and PrimerScript RT Reagent kits with gDNA Eraser (Perfect Real Time; Takara), respectively, according to manufacturer’s instructions, and stored at –20°C until further use.