FIG. 6.

RNAi-mediated Dicer knockdown. (A) Quantitative real-time RT-PCR analysis of the Dicer mRNA levels. The relative Dicer mRNA levels in siRNA-transfected cells were determined by normalizing the ratios of Dicer mRNA to β-actin mRNA against that of a nontransfected sample. An siRNA targeting the silkworm ACE gene was used as a negative control. The mean values and standard deviations from three independent experiments using the average value of triplicate RT-PCRs are shown. (B) Western blot analysis of the Dicer protein levels. Total cell lysates were resolved by 8% SDS-PAGE and detected with an anti-Dicer antibody. The levels of catalase are shown as a loading control. The indicated number (0.21) in the Dicer siRNA-transfected cells was determined by normalizing the ratio of Dicer to catalase against that of the control siRNA-transfected cells. (C) Dicer knockdown caused a defect in siRNA generation from shRNA precursors. Top, Western blot analysis of Dicer knockdown. Bottom, Northern blot analysis of the siRNA expression levels. Following transfection of pSilencer-lac operator into control and Dicer-depleted cells, small RNAs were detected by Northern blot analysis using radiolabeled probes homologous to the lac operator sequence. The levels of 5S rRNA stained with ethidium bromide are shown as a loading control. The indicated number (0.39) in the Dicer-depleted cells was determined by normalizing the ratio of siRNA to 5S rRNA against that of control cells.