Detection of Small RNA Molecules

Total RNA was extracted from N. benthamiana leaves infiltrated with A. tumefaciens, non-transgenic and transgenic soybean leaves, respectively, using the Trizol reagent (Cat no. 15596-026, Invitrogen, Carlsbad, CA, United States) following the instructions of the manufacturer. The RNA specimens were electrophoresed on 15% polyacrylamide/7 M urea gels and transferred to Hybond N⁺ membranes (Amersham) using a semidry blotter (Bio-rad, Hercules, CA, United States). The membranes were UV cross-linked in a HL-2000 HybriLinker™ (AnalytikJena: UVP) at 120,000 μJ/cm² energy for 4 min and separately hybridized with five different digoxin-UTP-labeled RNA probes. The mouse anti-digoxin monoclonal antibody (1:10000, Jackson ImmunoResearch) and IRDye 800CW-conjugated goat (polyclonal) anti-mouse IgG (1:15000; H + L; LI-COR Biosciences) secondary antibodies were used to evaluate siRNA production. The membranes were visualized using an LICOR Odyssey scanner with excitation at 700 and 800 nm. For Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assay, the 320 bp SMV CP and 1,812 bp GUS bands were amplified from pSMV-GUS-infiltrated N. benthamiana leaves. The Western blot analyses were performed according to previously described protocols and the size of CP protein is ∼ 30 kDa (Jiang et al., 2019 (link)).