Figure 5

Small lacZ gene-specific RNA is not present in the dsRNA preparation used to induce RNAi. RNA preparations from Drosophila cell cultures were fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad). Fractionated samples were analysed in 15% polyacrylamide-UREA gels, and electroblotted. A digoxigenin-labelled RNA probe complementary to the 5' region of the lacZ gene was used for the detection of siRNAs in lanes 1–3. Induction and detection of siRNAs to 35S promoter sequences in agroinfiltrated plant tissue was performed as described by Canto et al. (47) and were used as markers in lanes 4 and 5. The upper panel shows the small RNA species detected by probing with a lacZ gene-specific probe in DS2 cells transfected with pMT/V5-His/lacZ (lane 1) or non-transfected DS2 cells (lane 2) or in the dsRNA preparation used to transfect DS2 cells to induce RNAi (lane 3). Also shown are the small RNA species present in plant tissue agroinfiltrated with a binary vector (lane 5) or in non-agroinfiltrated tissue (lane 4) detected using a digoxigenin-labelled RNA probe to 35S promoter sequences present in the binary vector as markers. The positions of migrations of the small (~21nt) RNAs are indicated by arrows.