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. 2009 May;15(5):898–910. doi: 10.1261/rna.1268209

FIGURE 2.

FIGURE 2.

Dual gene silencing activity of linear tsiRNAs, tsiER(s25s21), tsiER(s21s21), and tsiER(s15s21) against EGFP and RLuc genes. (A) The predicted structure of tsiER RNAs transcribed from RNA Pol III promoters. (Boxes) Sites complementary to (green) EGFP and (red) RLuc target genes. In the pGD constructs, all colored guide/antisense strand sequences are located downstream from the U6 promoter. (Underlined) Probe sequences for detection of siE and siR antisense strands. (B) Luciferase assay of cellularly expressed tsiRNAs with sequential siE and siR components using the same direction of transcription. Transfection into Huh7 cells was performed as described in Figure 1C. The FLuc and RLuc expression levels of cells transfected with plasmid encoding control tsiRNA (tsiC) were set at 100%. Relative means (+SD) from three independent experiments are shown. (C) Northern blot analysis for detection of intracellularly processed siE and siR siRNAs from precursor tsiRNAs. Detection of cellular U6 snRNA was used as a loading control. (Arrows) Processed 21–23-nt antisense strand (AS) siRNA products. Probe sequences and positions are described in A. (Right) Marker sizes (in nucleotides, nt).