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Inducing RNAi with siRNA Cocktails Generated by RNase III

estion was run on a 15% non-denaturing acrylamide gel along with a 21 bp chemically synthesized siRNA to GAPDH, which served as a size marker. The gel was stained with ethidium bromide and photographed under UV light. 1B. RNase III derived siRNA cocktails silence GAPDH, La and c-FOS. GAPDH and La siRNA cocktails were transfected into HeLa cells. The c-fos siRNA mixture was transfected into 293 cells followed by 24 hours of stimulation with 50 nM PMA. All samples were harvested at 48 hours post transfection and immunofluorescence was performed with the appropriate antibodies. Fluorescence signal was quantitated, normalized for cell number and graphed.


Silencing by 12-15 bp RNase III Digestion Products. The size of chemically synthesized siRNA most often used for mediating RNAi is 21 bp (12). It has been shown that the 21 bp products generated by RNase III digestion are potent inhibitors of gene expression (10). However the products of a complete RNase III digestion are 12-15 bp. To compare the ability of these smaller products to reduce gene expression with 21 bp siRNA, a 200 bp GAPDH dsRNA was digested with RNase III under standard conditions and the resulting 12-15 bp fragments were acrylamide gel purified from the incomplete digestion products (Figure 2A). HeLa cells were transfected with 100 nM final concentration of the 12-15 bp purified products, as well as with the same concentration of a 21 bp chemically synthesized siRNA known to effectively reduce GAPDH levels. Figure 2B shows representative immunofluorescence images demonstrating reduction in GAPDH levels after transfection with the RNase III generated siRNAs. The 12-15 bp products are capable of reducing targ
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