FIGURE 2.

Presence of in vitro antisense Y-Sat transcript improves the detection of (−) strand Y-Sat siRNAs. (A,B) Detection of (+) strand (A) or (−) strand (B) Y-Sat siRNAs in the presence or absence of an in vitro–transcribed antisense (asY-Sat) or sense (sY-Sat) Y-Sat transcript. The amount of in vitro transcript added to each sample is given at the top of each lane. The “+” symbol indicates that RNA samples were extracted from Y-Sat-infected tobacco, while the “−” symbol indicates RNA from uninfected tobacco plants. (Lanes 1,10) In vitro transcripts only. The gray arrows indicate the position of siRNAs. (C) Detection of (−) strand Y-Sat siRNAs in the presence or absence of in vitro antisense Y-Sat transcript. The samples were treated differently prior to loading: (lane 1) total RNA dissolved in water and mixed with an equal volume of formamide loading buffer with no heating; (lanes 2–4) total RNA samples were dissolved in formamide, heated in boiling water for 5 min, and chilled on ice for 5 min; (lanes 5,6) total RNA samples were dissolved in water, boiled for 5 min, then mixed with an equal volume of 100% formamide loading buffer. Five micrograms of total RNA was loaded for each sample, except lanes 1 and 10 in A and B, which contain only the in vitro transcripts. (M) 21-nt radiolabeled RNA size marker.