Phenol (425 ml liquid phenol, 75 ml chloroform, 0.5 g 8-hydroxyquinoline)Procedure
Formate buffer - 0.15M sodium formate (10.3g/L), 0.15M formic acid (6ml/L)
Saturated ammonium sulfate - (optional), ammonium sulfate suppresses extraction of contaminants
0.2M NH4Ac (4.0 g NH4Ac, 250 ml DW)
Ethanolic NH4Ac (150 ml 0.2M NH4Ac, 28 ml absolute EtOH)
Sample solution: (for Laemmli gels) (Protein sample buffer (w\o reducing agent) + 10% Ficoll to make dense)
1- Grind 3 g tissue with 8 ml phenol, 12 ml formate buffer, (2ml saturated ammonium sulfate, optional) in a Waring blendor)
2- Centrifuge 5 min @ 7K in swinging bucket rotor (Sorvall HS4)
3- Withdraw 10 ml upper phase to fresh tube, add 2.2 ml absolute EtOH, swirl; add 280 mg CF11 cellulose, swirl twice to mix (incubation is unnecessary).
4- Centrifuge 5 min @ 6K, pour off supernatant. Suspend cellulose in 3 ml ethanolic NH4Ac, pipet into 3 ml syringe with cotton pad (pack with notched plunger). Run dry. Rinse remaining CF11 from tube with 2 ml ethanolic NH4Ac. Fill and drain column 2x with ethanolic NH4Ac. Add 0.75 ml 0.2M NH4Ac, drain.
5- Transfer column to fresh (Corex) tube; elute by filling once with 0.2M NH4Ac, drain. Add 6 ul 10% Ficoll, 8 ml acetone (or EtOH), mix, store at -20 C (10 min is plenty). Cloudy Ficoll precipitate should be visible.
6- Centrifuge 3 min @ 7K, pour off supernatant. Add 2 ml MeOH, translucent pellet becomes visible, swirl, pour off MeOH, invert tube to dry. Blow with hair drier to speed drying.
7- Dissolve pellet in 35 ul of sample buffer. Electrophorese 10 ul aliquot.
8- Run sample (overnight at ~160 V) on 8-16% polyacrylamide gradient gel (0.75 mm) and stain with silver. Scan with HP Scanjet; print with inkjet. Thin gels stain best. The concentrated gel survives handling well.
Features of dsRNA extraction procedure
- Some tissues contain substances that inhibit acidic phenol extraction of dsRNA. Salt facilitates extraction.
Without DNase treatment, DNA usually contaminates dsRNA, suggesting that it competes with dsRNA for adsorption to cellulose. Low salt and acid extraction reduce DNA recovery by phenol extraction (probably block histone removal). In six comparisons (different tissues), acid extraction always gave better dsRNA yields than neutral extraction (marginally better to several fold better). Yield was insensitive to moderate changes in pH or salt concentration.
Chloroform is an antifreeze and increases phase density so that insolubles stabilize the interphase. In a single comparison, 10:1 phenol:chloroform yielded more dsRNA than 1:1. With a 10:1 ratio insolubles occasionally appear in the pellet rather than the interphase. Octanol is an antifoam. Phenol extractions don't foam and require no octanol.
In early extractions, aqueous phases mixed with alcohol and CF11 were poured directly into columns. Viscous samples required hours to drain and wash. Pelleting and resuspending cellulose reduces column packing and washing time to roughly an hour for a dozen samples. CF11 can be pelleted immediately after mixing with the alcoholic phase.
My standard protein gels (8 to 20% gradient SDS PAG) work better than "dsRNA gels". Slightly lower acrylamide concentrations with less crosslinker in the upper solution works still better.
Ficoll is useful carrier. It makes precipitates and MeOH dried pellets visible. Ammonium acetate is self buffering (pH 7). Acetate salts are compatible with acetone (or isopropanol) precipitation.
Silver "staining" (about 5x as sensitive as ethidium bromide) produces a permanent image which can be cheaply scanned into an image file. I put gels into plastic sheet protectors, scan with an HP5200C (cheap), label the image with Paint Shop Pro and print the labeled image with an HP932C inkjet (cheap)
For most viruses dsRNA yield varies with tissue condition. Young, expanding, freshly infected tissue gives best yields. Older tissue yields less dsRNA. Extracting tissues varying in age is more likely to improve dsRNA yield than varying isolation technique. Some tissues contain substances (likely carbohydrates) which copurifiy with dsRNA (and stain with silver). In these cases fluorescent staining may be preferable. Sybr Gold (Molecular Probes) is the stain of choice.
Dodds. J.A. dsRNA in diagnosis. (1993) pp273-294 in Diagnosis of Plant Virus Diseases ed R.E.F Matthews, CRC Press, Boca Raton
Valverde, R.A., Nameth, S.T., Jordan, R.L. (1990) Analysis of Double Stranded RNA for Plant Virus Diagnosis Plant Dis74, 255-8.