Viral RNA Extraction

Summary

Viral RNA extraction can be (1) used for the study of RNA viral replication mechanisms, (2) used for reverse genetics studies, and (3) used for other studies in molecular virology.

Operation method

TE-saturated phenol/chloroform extraction method

Principle

Most of the plant viral RNA is single-stranded RNA, and its polarity is the same as that of mRNA. The extraction of plant viral RNA is relatively simple, and the use of phenol-chloroform can obtain satisfactory results.

Materials and Instruments

TMV Virus Solution
TE-Saturated Phenol:Chloroform Chloroform NaAc Ethanol TE Buffer Bacterial Water
Freezing tabletop centrifuge Low-temperature vacuum dryer Electrophoresis apparatus Electrophoresis tanks

Move

I. Preparation of experimental materials

1. Materials
Purification of TMV viral fluid (10 mg/ml).
2. Equipment
Frozen tabletop centrifuge, cryogenic vacuum dryer, electrophoresis instrument, electrophoresis tank.
3. Reagents
TE-saturated phenol:chloroform (1:1), chloroform, 3 M NaAc (pH 5.2), ethanol (100% and 70%), TE buffer, bicarbonate of water without RNAase.
II. Operational steps
1. Take an eppendorf tube and add 400 ml of purified TMV (10 mg/ml), then add an equal volume of phenol/chloroform, cap the tube tightly and shake it well by hand for 1 minute, then centrifuge it at 12,000 g at 4°C for 10 minutes.
2. Aspirate the aqueous phase into a new eppendorf tube and extract with phenol/chloroform until the interface between the aqueous and organic phases is free of protein.
3. Aspirate the aqueous phase into a new eppendorf tube, add an equal volume of chloroform, invert the tube by hand for several tens of seconds, and centrifuge at 12,000 g for 10 minutes at 4 °C.
4. Take the aqueous phase, add 1/10 times the volume of 3 mol/L NaAc (pH 5.2), 2.5 times the volume of ice-cold ethanol, mix well and leave at -20°C for 30 min.
5. Centrifuge at 12,000 g for 15 min at 4°C, carefully discard the supernatant, wash the precipitate with 70% ethanol and centrifuge at 12,000 g for 5 min at 4°C.
6. The supernatant is carefully discarded and the precipitate is dried under vacuum for 5 minutes or in air for 10 minutes and dissolved in RNAase-free bicarbonate of water or TE buffer.
7. Take 10 ml for electrophoretic analysis.

Caveat

1. The entire procedure should be carried out at as low a temperature as possible.

2. Since the viral RNA is embedded in the capsid proteins, multiple phenol/chloroform extractions are generally required to fully strip the viral capsid proteins.

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