Nucleic Acids Research, 2008, Vol. 36, No. 20 6519 Figure 4. Expression and eﬃcacy of perfectly complementary shRNA and lhRNAs. (A) Northern analyses of siRNA production in wild-type or mHelicase cells following transfection with perfectly complementary hairpin RNA constructs. Total RNA extracted from cells transfected with the empty vector was used as a negative control for cross-hybridization of each probe. The mature siRNA is indicated with the arrowhead and the precursor molecules are indicated by the vertical bars. The membrane was subsequently stripped and probed for miR-21. U6 snRNA expression was also detected as a loading standard. The same membrane was also stripped and probed for siRNA using Probe 2. The illustrated size marker is based on the migration of the Ambion Decade ladder. (B, C) The processing eﬃciency for each siRNA [Probe 1 (B) and Probe 2 (C)] was determined by autoradiography and dosimetry as follows: processing eﬃciency = matureIDV/(matureIDV + precursorIDV) IDV, Integrated Density Value. The mean and standard deviation of three experiments is represented. (D) Inhibition of reporter gene expression by the shRNA or lhRNA constructs. The sequence corresponding to either target 1 or 2 was inserted into the cloning site in the 30 UTR of the Renilla luciferase gene in the psiCHECK-2 plasmid. One hundred nanograms of each reporter construct and 1 ng of either SII-c21 or SII-50 hairpin RNA expression vector were co-transfected into wild-type or mHelicase cells. Luciferase values were determined 24 h later and the Renilla target was normalized to Fireﬂy luciferase, which is contained on the same reporter construct but is not subject to downregulation by the shRNA. Luciferase values in response to the empty vector (control) were set at 100%. P-values compare mHelicase versus wild type: target 1: SII-c21:P < 0.6; SII-c50:P < 0.001; target 2: SII-c50: P < 0.001. the G:U wobble pairs in the lhRNAs were not speciﬁc for the observed increase in processivity. Interestingly, we observed no appreciable diﬀerences in the processing eﬃ- ciency or siRNA accumulation from the SII-c21 vector in wild-type and Dicer mHelicase cells (Figure 4A and B), in contrast to the data obtained with G:U-containing shRNA, which showed reduced siRNA accumulation in vivo in the mHelicase cells. Furthermore, no signiﬁcant diﬀerences in target knockdown were observed in wild- type and Dicer mHelicase cells in response to the SII-c21 shRNA (Figure 4D, SII-c21, P < 0.6). These results are in contrast with the signiﬁcant reduction in knockdown eﬃ- cacy by the G:U wobble containing SII-21 shRNA in the Dicer mHelicase background, suggesting that shRNAs with thermodynamically stable stem regions are largely unaﬀected by the mHelicase insertion into hDicer. These results suggest that the thermodynamic stability of helical stem region of hairpin RNAs is an important feature for the Dicer helicase mutant phenotype and aﬀects the processing eﬃciency and siRNA accumulation in the Dicer mHelicase cells. Supporting this hypothesis is our observation that miRNAs with unpaired nucleotides