Gene silencing by RNA interference (RNAi) is a widely used approach for target-specific knockdown of gene expression. Induction of RNAi in mammalian cells can be achieved by introduction of synthetic small interfering RNA (siRNA) resulting in transient knockdown, or alternatively by stable expression of short hairpin RNA (shRNA). Several algorithms for efficient siRNA design exist, but recent reports have suggested that these cannot be directly used to design efficient shRNAs. In this study, 25 siRNAs targeting independent sequences in five transcripts were used for the construction of shRNA cassettes. Both the siRNAs and shRNA constructs were transfected into HEK293T cells. Quantitative real-time PCR analysis revealed that 19 out of the 25 shRNA constructs reduced the average expression level to less than 30%. Our data support that sequences designed by siRNA algorithms efficiently reduce the expression of the target gene when converted into shRNA expression constructs.