TITLE: Double stranded RNA extraction by cellulose
AUTHORS: Vahid Jalali Javaran

[DESCRIPTION]
In this protocol, the viral dsRNA extraction from infected-grapevine plants is explained.  

[STEPS]
SECTION: Total nucleic acid extraction
1. Weigh ~ 1.5 g of fresh or frozen leaves. Put the leaves in a 50 ml capped centrifuge tube containing 8X 8-mm stainless balls (sterilized). Immerse the tube in the liquid nitrogen for 5 mins. Rapidly mount the tubes in a foam holder and move to the MiniG chamber.
Fix the tubes correctly according to the instructions. Run at 1,500 rpm for 1 min. In the same way, prepare ~1 g of bean leaves (Phaseolus vulgaris) in a 50-ml capped centrifuge tube containing 8X 8-mm stainless balls and homogenize in the MiniG as mentioned above.
SECTION: Total nucleic acid extraction
2. Add 12 ml of extraction buffer and 120 µl of 2-mercaptoethanol to each sample, and mix well. Add 8 ml of extraction buffer and 80 µl of 2-mercaptoethanol to the bean tube, and mix well. 
SECTION: Total nucleic acid extraction
3. Move 120 µl from bean tissue suspension in each sample. shake for 40 mins at 300 rpm and centrifuge at 1000 x g for 1 min at 10◦C to remove bubbles and a large amount of debris. Decant supernatant to a new 50-ml tube.
SECTION: Total nucleic acid extraction
4. Add 12 ml of 5.8 M potassium acetate to the supernatant, mix thoroughly and centrifuge at 14,000 x g for 15 mins at 10◦C.
SECTION: Total nucleic acid extraction
5. Decant the supernatant through 3 layers of sterilized cheesecloth (optional) into a clean 50 ml centrifuge tube, and add 16 ml of 100% isopropanol. Mix. Leave at -20C for 20 min. (safe pause point)
SECTION: dsRNA purification by cellulose
7. Resuspend the pellet in 20 ml STE-18, vortex. Centrifuge at 14,000 x g for 15 min at 4◦C. Decent to a new 50-ml centrifuge tube. 
SECTION: Total nucleic acid extraction
None. 
SECTION: dsRNA purification by cellulose
8. Add 2 ml  Sigmacell cellulose suspension (0.3 g) and vortex. Shake at 300 rpm for 15 min at room temperature. Centrifuge tubes at 14,000 x g for 5 min at 20◦C and discard the supernatant.
SECTION: dsRNA purification by cellulose
9. Resuspend the pellet in 40 ml STE-18. Centrifuge at 14,000 x g for 5 min at 20◦C and discard the supernatant. Repeat this step once by adding 20 ml STE-18 to suspend the pellet and centrifuge at 14,000 x g for 5 min at 20◦C. Discard the supernatant.
SECTION: Total nucleic acid extraction
6. Centrifuge at 11,000 x g for 16 mins at 4◦C. Carefully discard the supernatant.
SECTION: dsRNA purification by cellulose
10. Evaporate ethanol from cellulose pellet at 40◦C for 15 min. Resuspend the pellet in 6 ml 1×STE. Shake at 300 rpm for 15 min at RT (room temperature). Centrifuge at 14,000 x g for 8 min at 20◦C. Pour the supernatant into a new 50-ml centrifuge tube (it is still ok even with some cellulose particles).
SECTION: dsRNA precipitation
11. Add 0.6 ml 3M NaOAc and 12 ml anhydrous alcohol. Mix well and leave at -20◦C for 20 min. (safe pause point)
SECTION: dsRNA precipitation
12. Centrifuge at 11000 x g for 15 min at 4◦C. Carefully discard the supernatant. Rinse the pellet with cold 70% Ethanol twice (if the pellet detached, centrifuge again). Air dry. Resuspend the pellet in 300 µl TE or sterile water, and transfer the suspension in a 0.85 um PES filter column (Sartorius Stedim Biotech), centrifuge at 13,000 x g for 30 seconds at RT. Use 100 µl TE or water to rinse again the tube and transfer the liquid to the same filter column and centrifuge for 2 mins. The total volume is 400 µl. Discards the filter. Store a -80◦C (safe pause point) or proceed to digestion.