FIGURE 4.
Silencing ability of species randomly selected from the TNF-specific siRNA library produced by the Dicer method. (A) 293FT cells were cotransfected with randomly chosen clones, a TNF expression vector, and pSEAP using Lipofectamine 2000 (Invitrogen). TNF was assayed by ELISA and SEAP by a colorimetric assay 48 h post-transfection. The inhibition by each siRNA is shown, normalized to the SEAP control target. Rationally designed control shRNAs targeting TNF (shRNA-TNF-229) and DsRed (shRNA-DsRed-2) were expressed from pU6. Rationally designed control siRNAs targeting TNF (siRNA-TNF-229) and DsRed (siRNA-DsRed-2) were expressed from pU6/H1. (B) Representative sequences of the assayed clones were classified into three groups depending on reduction in TNF expression: effective (>60% inhibition), intermediate (20%–60%), and ineffective (<20%).