Testing the limitations of miRNA-embedded shRNAs and multiple U6/shRNA cassettes by inhibition of Luciferase reporters. (A) The left panel shows a schematic representation of the predicted transcription products of a standard 19-nt shRNA (shRNA-19nt), a miR-30 mimic shRNA with a 3-nt extended stem and mismatched bp in the passenger strand (shRNA-22nt), a miRNA mimic shRNA from the first locus in pRFPRNAi (p1-miRNA), and a miRNA mimic shRNA from the second locus in pRFPRNAi (p2-miRNA). For each the siRNA stem is shown in red. The right panel shows normalised ratios of the Renilla:Firefly luciferase activity when DF-1 cells were co-transfected with the indicated RNAi plasmids and their target Luciferase reporter plasmids (asterisks indicate P < 0.05 compared to equivalent 19-nt regular shRNAs, n = 4, two-tailed unpaired T-tests). (B) The top panel shows a schematic representation of the multiple U6/shRNA constructs featuring 1, 2, 3, 4 or 5 individual U6/shRNA cassettes. The bottom panel shows normalised ratios of the Renilla:Firefly luciferase activity when DF-1 cells were co-transfected with the indicated RNAi plasmids and each of the luciferase reporter plasmids. The +VE control shRNAs used for each reporter were: sh-1a for psi-CHK-1, sh-2 for psi-CHK-2, sh-3 for psi-CHK-3, sh-4 for psi-CHK-4 and sh-5 for psi-CHK-5. Values are shown as percentages of the negative control shRNA (sh-NS), as the mean of 4 replicates ± standard error.