An Optimized/Scale Up-Ready Protocol for Extraction of Bacterially Produced dsRNA at Good Yield and Low Costs

Double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) and lead to directed silencing of specific genes. This natural defense mechanism and RNA-based products have been explored for their potential as a sustainable and ecofriendly alternative for pest control of species of agricultural importance and disease vectors. Yet, further research, development of new products and possible applications require a cost-efficient production of dsRNA. In vivo transcription of dsRNA in bacterial cells has been widely used as a versatile and inducible system for production of dsRNA combined with a purification step required to extract the dsRNA. Here, we optimized an acidic phenol-based protocol for extraction of bacterially produced dsRNA at low cost and good yield. In this protocol, bacterial cells are efficiently lysed, with no viable bacterial cells present in the downstream steps of the purification. Furthermore, we performed a comparative dsRNA quality and yield assessment of our optimized protocol and other protocols available in the literature and confirmed the cost-efficiency of our optimized protocol by comparing the cost of extraction and yields of each extraction method.

Automated summary

Double-stranded RNA (dsRNA) is a natural defense mechanism that can silence specific genes through RNA interference (RNAi). It has potential as a sustainable and ecofriendly alternative for pest control and disease vector management. However, cost-efficient production of dsRNA is needed for further research and development. In this study, we optimized an acidic phenol-based protocol for low-cost extraction of dsRNA from bacteria, with high yield and no viable bacterial cells remaining in the purified dsRNA. Comparative assessments confirmed the cost-efficiency of our optimized protocol.