Luciferase-based transient co-transfection experiments to compare shRNA and miRNA design schemes. Knockdown studies were performed in duplicate as co-transfections of shRNA:pGL3-gene:pRL at the indicated ratio. Firefly luciferase values are divided by renilla values to control for transfection efficiency, and all knockdown data are normalized to control hairpins. (A) Comparison of published modifications to standard H1-shRNA design (F = frayed, miR23 = loop derived from miR-23, and N19 = standard pSuper shRNA design) directed against the pGL3-MELK reporter. Cells were transfected with a 10:1 ratio of the indicated shRNA to pGL3-Melk reporter. (B) CMV-miR to H1-shRNA conversion scheme. The active 'siRNA' was identified within the pol II-miRNA and converted to a standard H1-shRNA hairpin (black bars = Drosha and Dicer processing; red = guide strand, blue = miR-155 loop, pink = pSuper loop). (C) Comparison of H1-shRNA and CMV-miR vectors corresponding to murine p53 sequences transfected at 7.5:1, 5:1 and 2.5:1 (shRNA:pGL3-mu-p53). (D) The removal of the EmGFP leader renders miR-p53-1 non-functional at a co-transfection ratio of 10:1.