Targeting cellular genes with PCR cassettes expressing short interfering RNAs

The synthesis and transfection of PCR short interfering/short hairpin RNA (si/shRNA) expression cassettes described in this chapter can be used to rapidly test siRNA targeting and function in cells. One critical element in the design of effective siRNAs is the selection of siRNA-target sequence combinations that yield the best inhibitory activity. This can be accomplished by using synthetic siRNAs and transfection procedures, but these can be costly and time consuming. By using the PCR strategy, it is possible to create several expression cassettes and simultaneously screen for the best target sites on any given mRNA. This PCR strategy allows a rapid and inexpensive approach for intracellular expression of si/shRNAs and subsequent testing of target site sensitivity to downregulation by RNA interference (RNAi).