N membrane

Twenty μg of denatured total RNA was loaded onto a 15% polyacrylamide TBE gel and separated in a 1 X TBE running buffer, followed by transfer onto a N⁺ membrane (Amersham, London, UK) at 200 mA for two hours in an electro-transferring system and crosslinking under ultraviolet radiation for 150 seconds. The miRNA-specific oligo was 5’ end labelled with γ-³²P-ATP through T4 polynucleotide kinase (Takara), according to the manufacturer’s protocol. The oligo probes were designed based on individual miRNA sequence information deposited in miRBase (http://microrna.sanger.ac.uk). An antisense oligo of U6 snRNA was used to detect U6 snRNA from each sample as a loading control. After prehybridisation using hybridizing buffer (BioDev, BJ, China), blots were hybridized with ³²P-labelled DNA probes (2 μmol/ml) overnight at 37°C. After washing, the hybridized membranes were exposed to Kodak X-omat BT film.

The protocol established by E. M. Southern for Southern blot analysis was used to confirm the transformation events. Total genomic DNA (5 μg) of Arabidopsis seedlings was digested with EcoR I-HF or Hind III-HF (New England Biolabs) overnight at 37 °C, fragments were separated by gel electrophoresis, transferred to N+ membrane (Amersham Biosciences, Roosendaal, The Netherlands) using the capillary transfer method, hybridized with a radioactive isotopes [α-32P] dCTP-labeled cDNA probe specific for nt 5,161 to 5,620 of the 3′ BrYV fragment, recorded by phosphor autoradiography, and finally scanned using a Typhoon 9000 (GE Healthcare). Primers used to amplify the probes are listed in Table S5.