A new method for extraction of double-stranded RNA from plants

Area of Science:

• Plant pathology
• Molecular biology
• Virology

Background:

• High molecular weight double-stranded RNA (dsRNA) in plants indicates RNA virus presence.
• DsRNA is a stable molecule, easily extracted and valuable for characterizing novel, hard-to-purify viruses.
• Existing dsRNA purification protocols often rely on hazardous organic solvents like phenol and chloroform.

Purpose of the Study:

• To develop a novel dsRNA extraction protocol using a lithium salts buffer.
• To provide an alternative to organic solvent-based methods for dsRNA purification.
• To enable efficient dsRNA extraction from recalcitrant plant species, including Vaccinium hosts.

Main Methods:

• Developed a dsRNA extraction protocol utilizing a lithium salts buffer.
• Avoided the use of organic solvents, except for alcohols, in the extraction process.
• Validated the protocol on various plant species, including challenging Vaccinium hosts.

Main Results:

• The lithium salt buffer method yields comparable dsRNA amounts to traditional protocols.
• Consistent dsRNA recovery was achieved from Vaccinium hosts previously resistant to purification.
• Purified dsRNA quality is suitable for downstream enzymatic applications, such as reverse transcription-polymerase chain reaction and cloning.

Conclusions:

• The developed lithium salt buffer protocol offers an effective and safer alternative for dsRNA extraction.
• This method enhances the ability to study RNA viruses in a wider range of plant species.
• The protocol facilitates further molecular analysis and characterization of plant viruses.