Evaluation of RNA isolation techniques for enhanced dsRNA yield and purity in bacterial expression platforms

Abstract

RNA interference (RNAi) in pest control is emerging as a targeted, eco-friendly alternative to chemical pesticides in agriculture. Efficient and cost-effective production of high-yield and high-purity dsRNA is crucial for the success of this approach. Escherichia coli HT115 (DE3), commonly employed for dsRNA synthesis, offers high transformation efficiency and genetic flexibility. However, the yield and quality of dsRNA are strongly influenced by the RNA isolation and purification protocols used. The present study compares six RNA isolation methods, viz. TRIzol-isopropanol, TRIzol-absolute ethanol, RNA-XPress-isopropanol, RNA-XPress-absolute ethanol, ethanol isolation, and extended ethanol precipitation using the E. coli-L4440 expression system. The TRIzol-absolute ethanol method yielded the highest total RNA concentration (5.27 mg/mL), followed by TRIzol-isopropanol (4.84 mg/mL). Although ethanol isolation (1.35 mg/mL) and extended ethanol precipitation (1.87 mg/mL) produced lower total RNA quantities, they demonstrated superior dsRNA recovery efficiencies up to 84.44%. The findings underscore the importance of selecting and optimizing RNA isolation protocols to maximize dsRNA yield and purity, which are crucial factors for advancing RNAi-based pest management strategies. The results offer practical guidance for scalable dsRNA production, contributing to the development of sustainable agricultural practices.