Testing plant-derived siRNAs in mammalian cells. (A) Testing plant siRNAs for gene silencing. Gene silencing assays were performed in 96 well plate format. Cells were co-transfected with pmC9-dsEGFP and pdsEGFP (70 ng each) together with 18 μg of total Tc9 RNA (Tc9), 10 μg of total tomato RNA (Tom), gel purified Tc9 small RNAs (Tc9p) contributing 5 nM Tc9si or tRNA (amount indicated in ng) using Lipofectamine 2000 (Lipo2000), Code Breaker (CB) or RNAiFect (RF) transfection reagents. (B) Effect of KOAc and PVP treatment. Left Hand Side. Silencing assays contained 3 ng of gel-purified Dc9si and Tc9 small RNAs containing 3 ng of Tc9si that had been pre-treated (or not) with 200 mM KOAc (see materials and methods). Right Hand Side. Assays were performed with 3 ng of KOAc treated siRNA samples in the presence and absence of 2% Polyvinylpyrrolidone (PVP) (see materials and methods). (C) Effect of other small RNAs on gene silencing. Left Hand Side. Silencing assays were performed with increasing amounts (indicated in ng) of gel-purified small RNAs (TsRNAs) from wt tobacco plants together with Dc9si. Right Hand Side. Cells were co-transfected with pdsEGFP only and one amount of Esi (12 ng) together with increasing amounts of a synthetic GAPDH siRNA (Gsi). (D) Effect of plant small RNA preparation protocol on Dc9si activity. Dc9si was mixed into the tobacco wt plant sample (at 50 ng/g of leaf) during the RNA extraction process. Extracted RNAs were gel purified and recovered Dc9si (Dc9si*) was quantitated by RPA (right panel). For silencing assays, cells were co-transfected with pmC9-dsEGFP and pdsEGFP with 3 ng of Dc9si or 3 ng of two different preparations of Dc9si*. (E) Effect of RNase digestion and serum treatment on siRNAs. Left hand side. Unlabeled Dc9si and 32P-body-labeled Dc9si (BL) were treated (treated (+) or untreated (-)) with a limited amount of RNAses as described in materials and methods. Dc9si was detected by RPA and BL Dc9si was detected directly on the same gel. 32P-end labeled synthetic RNA oligonucleotides of 25 and 21 nt are size markers. For serum treatment (right hand side), Dc9si and Tc9si were treated with FBS (see materials and methods) for the indicated times. FBS treated products were detected by RPA.