Fig. 8. Detection of small RNAs in silenced plants. Total RNA from the indicated plants was prepared using a procedure to enrich for small RNAs (Hamilton and Baulcombe, 1999). This RNA was electrophoresed on 15% acrylamide gels, electroblotted onto a nylon membrane, and probed with NOSpro sense (S) and antisense (AS) RNA probes. (A) Small NOSpro sense (lane 5) and antisense (lane 9) RNAs ranging in size from 23 to 25 nt were detected only in silenced plants. These small RNAs were not present in the original NOSpro target line (lanes 3 and 7), in non-silenced plants that synthesized polyadenylated single-stranded NOSpro RNA (lanes 4 and 8), or in silenced plants following the introduction of the 271 locus (lanes 6 and 10), which disrupted synthesis of NOSpro dsRNA (Figure 2). As size and polarity controls, NOSpro sense (lanes 2 and 12) and antisense (lanes 1 and 11) DNA 23mers were used. The larger band visible in lane 2 is probably a multimer of the oligonucleotide present in the commercial preparation. Larger bands in lanes 7–10 are due to non-specific hybridization of the RNA probe. (B) Detection of NOSpro small RNAs in plants containing a transcribed NOSpro IR created in planta by Cre recombinase (lanes 1–4). NOSpro small RNAs were not detected in plants containing a transcribed NOSpro DR before conversion by Cre recombinase (lanes 5–8). Identical results were obtained with a NOSpro-S probe (not shown). The eight plants shown here are the same as those shown in Figure 5, panels 1–8. AS and S lanes show control antisense and sense DNA 23mers, respectively.