Synthesize an a 32P-labeled in vitro transcript complimentary to the target RNA , and an unlabeled transcript of the same sense as your target RNA (see In Vitro Transcription protocol # 28)
1 ml Buffer: 853 µl deionized Formamide
85 µl 5 M NaCl
2 µl 0.5 M EDTA
64 µl 0.5 M PIPES:KOH, pH 6.5
Combine 1 µl probe (10,000 cpm)
1 µl viral RNA (~200 ng) (or, 2 µg total RNA from infected plant)
30 µl Annealing buffer
Incubate at 85oC for 2 min. then at 50-55oC for 2 to 3 hours
Chill on ice: 920 µl water
60 µl 5 M NaCl
10 µl 1 M Tris pH 7.5
10 µl 0.5 M EDTA
2 µl RNase A (Sigma, 10 mg/ml)
2 µl RNase T1 (Calbiochem 2.5 Units/µl)
Quick cool RNA samples on ice. Add 300 µl digestion mix to each sample.
Incubate at 15oC (also works at 30oC) for 1 hour. Add 20 µl 10% SDS, &
10 µl Pronase E (10 mg/ml) and incubate at 37oC for 15 minutes.
Add 5-10 µg carrier tRNA, extract with phenol:chloroform, and add 40 µl
3 M NaOAc and 1 ml of ethanol to aqueous phase. Incubate at –20oC.
Analyze protected fragments by denaturing gel electrophoresis, either agarose (for larger fragments) or Urea/acrylamide.
Notes: Always use an unlabeled (+) transcript control to distinguish mismatches from A-U rich breathing.
See: Owen & Palukaitis, Virology 166:495, & Roossinck and Palukaitis, Mol.
Plant Mic. Interact 3:188.