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)

Annealing

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 85^oC for 2 min. then at 50-55^oC for 2 to 3 hours

 

RNase Digestion

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 15^oC (also works at 30^oC) for 1 hour. Add 20 µl 10% SDS, &

10 µl Pronase E (10 mg/ml) and incubate at 37^oC 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 –20^oC.

 

Gel Electrophoresis

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.