RNA-RNA interaction and electrophoretic mobility shift assay (EMSA)

For RNA-RNA interactions of EHDV RNA segments, 150 ng linearized plasmid of each segments (S5-S10) were transcribed either in pairs or combinations of multiple segments. RNA transcription was carried out in a buffer containing 40mM Tris, pH7.5; 10mM MgCl₂; 20mM NaCl₂, 3mM spermidine, 50mM DTT; 5mM each rNTPs; 10U RNase inhibitor and 40U of T7 RNA polymerase (Thermo Scientific) for 3 h at 37°C followed by RNase free DNase 1 treatment. Immediately after DNase treatment, detection of RNA complexes was done by electrophoretic mobility shift assay (EMSA). Electrophoresis gel was run for 180 min at 150 V in TBM buffer (45mM Tris, pH8.3; 43mM boric acid; 0.1mM MgCl₂) and stained with 0.01% (w/v) ethidium bromide. The integrity of transcribed RNA was checked routinely by denaturing gel electrophoresis.