Principles of dsRNA extraction
dsRNA extraction principles
Plant viruses are mostly RNA viruses. DsRNA generally accompanies RNA virus replication. Whether dsRNA is a replication intermediate or a dead end product is not yet clear. Viruses produce dsRNAs corresponding to genomic and subgenomic RNAs. Consequently infected tissues contain virus-specific dsRNAs. Since healthy plants produce little or no dsRNA, purified dsRNA is useful for diagnosis.
Richard Franklin in the 1960s developed a method for purifying phage dsRNA. This involves extracting total nucleic acids, adsorbing dsRNAs to granular cellulose at low ethanol concentration and then eluting dsRNAs with ethanol free buffer. Nucleic acid purification from plant tissue is notoriously problematic because plants are full of carbohydrates which copurify with nucleic acids (phenol extraction was initially a carbohydrate purification procedure). Morris and Dodds found that the Franklin procedure was wonderfully selective for dsRNA and useful for diagnosing plant viruses.
When I began using "Morris and Dodds" I tested several tricks and encountered several problems. Firstly I substituted ammonium acetate for TBE buffer. Secondly I adopted Ficoll as a carrier for dsRNA precipitation. Thirdly I precipitated RNA with acetone (compatible with ammonium acetate). Fourthly I realized that acidic phenol would reduce extraction of chromosomal DNA and ssRNA (and probably carboxylate containing carbohydrates). Getting the right conditions proved challenging. Moderate salt concentrations (not pure acetic acid) were necessary for "complete" extraction and strongly acid conditions (formate as opposed to acetate buffers) were appropriately selective. In the process I found that ammonium sulfate suppressed extraction of some host contaminants (and facilitated extraction of others). Finally I found that extracts with added ethanol were often viscous, making columns slow. Centrifuging cellulose from ethanol containing extracts and resuspending in a small volume of ethanolic ammonium acetate greatly speeded columns. I often extract 12 samples in an afternoon, load a gel (polyacrylamide, ~10 to 20% gradient, low crosslinker in the 10% solution) before I go home and stain it the following morning.
I resolve dsRNAs by polyacrylamide gel electrophoresis and stain with silver. Agarose has poor resolving power and a high fluorescence background. Silver staining is more sensitive than ethidium bromide, produces a permanent record and can be scanned by cheap flatbed scanners. If you prefer fluorescence, be aware that Sybr Gold (Molecular Probes) is vastly superior to ethidium bromide.
The procedure works poorly for RNAs with genome linked proteins. Perhaps digesting away the protein would help. Let me know if you solve the problem.
Young leaves are better than old leaves (example)
Varying buffer concentration between 0.05 and 0.50M formate did not affect extraction
Increasing ammonium sulfate concentration increases extraction of some host components and decreases extraction of others. Ammonium sulfate is a precipitant, but dsRNA is highly soluble and unlikely to precipitate. Ammonium sulfate probably increases adsorption of components to cellulose in ethanolic ammonium chloride.
Adding ethanol to ammonium sulfate containing extracts gives a precipitate with dissolves on mixing.
A few plants have large amounts of material that copurifies with dsRNA. On occasion I pour supernatants from pelleting cellulose into a beaker. Some supernatants precipitate when mixed. Clearly plant species differ and some are better for dsRNA extraction.
Presumably more acidic acetate buffers would work as well as formate.