FIGURE 6.

sshRNAs monomers are not Dicer substrates. Eight picomoles of each synthetic shRNA (after heating and snap-cooling) were incubated in a 10 μL reaction in the presence of 1 U of recombinant Dicer enzyme (Stratagene) and buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 8), and 2.5 mM MgCl₂ for 18 h at 37°C. Control reactions that contained each shRNA but lacked Dicer were incubated in parallel. Samples were analyzed by (A) nondenaturing 10% PAGE and (B) denaturing 12% PAGE (8 M urea/20% formamide) and were stained with SYBR Gold. Note that SYBR Gold did not stain single-strand RNA as strongly as double-strand RNA.