As illustrated in Figure 1.3 of Chapter 1, the basic procedure of a cell-based genome-scale RNAi screen includes selection of an RNAi library to be screened, choice of human cells, transfection of siRNA into cells, treatment or incubation, detection, and statistical analysis. Following this procedure, the success of a cell-based genome-scale RNAi screen in this procedure relies on the design of the following elements: siRNA, control, plate, sample size, and methods for optimizing siRNA delivery and transfection efficiency. All these designs are explored in this chapter.

Beginning with this chapter, most of the work discussed in this book focuses on RNAi screens using siRNA; however, the designs and methods described in the following chapters are also applicable to RNAi screens performed in microplates with other silencing reagents, including shRNA, esiRNA, and dsRNA, which are described in Chapter 1.

siRNA Designs

The first step in starting a genome-scale RNAi screen is to choose an RNAi library. One important criterion for an siRNA library is well-designed siRNAs. siRNA design is critical for both successful gene knockdown and on-target hit selection. The quality of siRNA design is gauged mainly by the potency and specificity of the siRNAs in a library. siRNA design is sometimes performed by commercial siRNA vendors. In other cases, researchers also must get involved in siRNA design by either doing it alone or cooperating with the vendors.