Silencing by RNase III Digested dsRNA. We next analyzed the silencing ability of the RNase III generated siRNA cocktails. GAPDH and La proteins in HeLa cells are abundant and endogenous levels are easily detected. However the endogenous level of c-FOS in 293 cells is relatively low, and reduction in protein levels makes the protein undetectable. In order to overcome this limitation, 293 cells were stimulated to increase c-FOS protein levels by the addition of 50 nM phorbol ester (PMA) for 24 hours prior to protein analysis. RNase III-generated siRNA cocktails to GAPDH and La were transfected into HeLa cells, and the c-fos siRNA population was transfected into 293 cells. Immunofluorescence was used to examine the gene silencing effect. The fluorescent signal from this experiment was then quantitated and normalized for cell number. Figure 1B shows that protein levels were reduced by 78% for GAPDH, 86% for La, and 75% for c-FOS by introduction of the respective siRNA cocktails. These data demonstrate that RNase III generated siRNAs are very efficient at reducing target gene expression.
Figure 1. Silencing Gene Targets by RNase III
Derived siRNA Cocktails. A 200 bp dsRNA (15 g) for each gene
of interest was digested with 2.5 U RNase III for 1 hour at 37C. 1A.
RNase III efficiently digests dsRNA. One microgram of the dsRNA before
and after RNase III dig