RNA extraction and Northern Blot
The total RNAs for northern blot were extracted using hot phenol
method (Kohrer and Domdey, 1991). Then equal amount (15 μg) of RNAs were
used for electrophoresis and transferred to nylon membranes. Digoxigenin
(DIG)-labeled probes targeting the CP-coding sequence of ApNMV were
generated and used for hybridization to detect the positive-strand RNA3
((+)RNA3) and (+)RNA4 per manufacture’s protocol (Roche). Anti-DIG
antibody (conjugated with alkaline phosphatase, Roche) was used for
immunoblotting and a chemiluminescent substrate for alkaline phosphatase
was applied for imaging through a gel imager (Bio-RAD).