Enzymatic Production of RNAi Libraries from cDNAs and High-Throughput Selection of Effective shRNA Expression Constructs

RNA interference (RNAi) using small interfering (siRNA) or short hairpin RNA (shRNA) has become the first choice for gene silencing maneuver in mammalian cells. Because different siRNAs of the same gene have variable silencing efficacy and only limited siRNAs are functional, many candidates are necessary to identify optimal siRNAs. We have previously reported a method named e nzymatic p roduction of R NAi l ibrary (EPRIL), by which a great variety of shRNA expression constructs (RNAi library) can be produced simultaneously from cDNAs of interest. Recently, we have improved this method and developed a more efficient method. We describe in this chapter detailed protocols for the improved version of EPRIL and high-throughput selection of effective shRNA expression constructs from an RNAi library.