6516 Nucleic Acids Research, 2008, Vol. 36, No. 20 Figure 2. Expression and eﬃcacy of shRNA or lhRNAs in vivo. (A) Schematic representation of short and long hairpin constructs. Predicted structures of shRNA and lhRNAs, which target the HIV-1 rev exon (site II or SII) and the relative positioning of the oligonucleotide probes for detection of processed siRNAs are shown. The bases shown in the top strand indicate altered nucleotides in the passenger stand of the duplex, which create G:U wobble base parings within the sh- or lhRNA but do not aﬀect the perfect complementarity of the guide strand with its target. (B) Northern analyses of siRNA production in wild-type or mHelicase cells transfected with each hairpin RNA expression vector. 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 respective 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 siRNAs using Probe 2 and Probe 3. The illustrated size marker is based on the migration of the Ambion Decade ladder. (C) The processing eﬃciency for each hairpin RNA (Probe 1, Probe 2, Probe 3) 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.