Figure 4

Test of different hairpin designs reveals most efficient processing of small RNA from short hairpins based on the miR-122 structure. a. Sequence of different short hairpin RNAs expressed from the inducible pNEBR-X1 vector. miR122 stem-loop (blue); surrounding genomic DNA sequence (black); MCS of the vector (gray lines); a short hairpin based on miR-30 (green), carrying a new guide strand sequence (red). b. Northern blots of resulting short RNAs from the short hairpin plasmids described in (a) or empty vector. The plasmids were transfected into NIH3T3-47 cells, which were treated with 0.5 μM RSL1 or DMSO for 48 h. Short hairpins were detected with probes complementary to the guide strand for Sh-1, -2, -3 or miR-122, respectively; U6 hybridization was used as loading control. (See Methods for probe sequences.)