In Vitro Method for Synthesis of Large-Scale dsRNA Molecule as a Novel Plant Protection Strategy

Double-stranded RNA (dsRNAs) molecules are the precursors and effective triggers of RNAi in most organisms. RNAi can be induced by the direct introduction of dsRNAs in plants, fungi, insects, and nematodes. Until now RNAi is usually established by transformation of the plant with a construct that produces hairpin RNAs. Alternatively, advances in RNA biology demonstrated efficiently the in vitro method of large-scale synthesis of dsRNA molecule. Here we describe the de novo synthesis of dsRNA molecule targeting the specific gene of interest for functional application. Selection of off-target effective siRNA regions, flanking of T7 promoter sequences, T7 polymerase reaction, and maintenance of the stability of dsRNA molecules are the main criteria of this method to obtain pure and effective yield for functional applications. IPTG (isopropyl-β-D-thiogalactopyranoside) induced, T7 express E. coli cells, could be used for large scale synthesis of dsRNA molecule are also described in this method.