Detecting miRNA408 in Sweet Potato Leaves

Total RNA (20 µg) extracted from sweet potato leaves was separated by electrophoresis using a 12% polyacrylamide gel containing 8 M urea (Amresco Inc., USA). Then the RNA gel was transferred to a Hybond-NX membrane (GE Healthcare, USA) and cross-linked by UV (Pall et al., 2007 (link)). For miR408 detection, the blotted membranes were hybridized with the radiolabeled gene-specific RNA probes, produced by in vitro transcription (Jeng et al., 1990 (link), 1992 (link)) using T3 RNA polymerase (Promega, Madison, WI, USA). The antisense sequence of miR408 fused with the T3 promoter was synthesized and annealed with the T3 top strand (Supplementary Table S2) as the DNA template for transcription to synthesize the miR408 RNA probe by T3 RNA polymerase (Promega). The procedures of pre-hybridization, hybridization, and washing were performed as previously described (Lin et al., 2012 (link)). The membrane was exposed to a Phosphorimager screen (Molecular Dynamics) for 3–4 d after washing, and then was scanned by Phosphorimager (Typhoon 9400). In addition, the membrane was stripped and re-hybridized with the radiolabeled 5.8S rRNA probe, produced by PCR with primers 5.8S rRNA-F/5.8S rRNA-R (Supplementary Table S2), and it served as an internal control for small RNA blot assays. Three independent experiments were performed for each sample.