Mar 06, 2026
Isolation of dsRNA from Plants by Cellulose Chromatography
- Marta Niedzicka1,
- Stephen Byrne1
- 1Teagasc, Crop Science Department
Protocol Citation: Marta Niedzicka, Stephen Byrne 2026. Isolation of dsRNA from Plants by Cellulose Chromatography. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2q524l1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2024
Last Modified: March 06, 2026
Protocol Integer ID: 97491
Keywords: plant viruses, dsRNA, cellulose chromatography, dsrna from plant, isolation of plant virus, plant virus, dsrna, cellulose chromatography this protocol, plant, isolation
Funders Acknowledgements:
Horizon Europe - Marie Sklodowska-Curie Actions
Grant ID: 101106728
Disclaimer
Adjusted protocol from Luan, Y., Fan, W., Korxeelor, X., & Wu, X. (2024). Isolation of dsRNA from Plants by Cellulose Chromatography. In Double-Stranded RNA: Methods and Protocols (pp. 13-17). New York, NY: Springer US.
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Abstract
This protocol describes the process The protocol for isolation of plant viruses based on dsRNA adapted from Luan et al., 2024 (see disclaimer or references for full citation).
Materials
Equipment
1. Precision analytical digital lab scale
2. Refrigerated Centrifuge
3. Horizontal shaker
4. Pipettes
5. Biosafety cabinet
6. Centrifuge tubes 1.5 mL and 2 mL
7. Fume hood
8. Qubit
Chemical and reagents
1. Liquid nitrogen
2. CF11 cellulose (Whatman) - replaced by C6288 cellulose by Sigma
3. Ethanol
4. Polyvinylpolypyrrolidone (PVP-40, Sigma-Aldrich)
5. Tris-HCl
6. EDTA
7. Molecular biology graded water
8. NaCl
9. SDS
10. β-mercaptoethanol
11. Sodium acetate
12. Isopropanol
Buffers
1. 10 % volume SDS
SDS precipitates at low temperatures. The solution should be prepared with heating and needs to be warmed to at least room temperature prior to use.
2. 1 M Tris-HCl (8.0 )
3. 0.5 M EDTA (8.0 )
EDTA is difficult to dissolve at high pH; therefore, it is necessary to measure the pH of the solution when adding NaOH to adjust the pH.
4. 10 % volume PVP-40
5. Nucleic acid extraction (NAE) buffer:50 millimolar (mM) Tris-HCl (8.5 ), 50 millimolar (mM) EDTA, 3 % volume SDS, 1 % volume β-mercaptoethanol, 1 % volume PVPP-40
6. 10×dsRNA-binding (10×DSB) buffer: 0.1 M Tris-HCl (8.0 ), 1 Molarity (M) NaCl, 0.01 Molarity (M) EDTA
7. 1×DSB buffer (containing 20% Ethanol): 10 mM Tris-HCl (8.0 ), 100 millimolar (mM) NaCl, 1 millimolar (mM) EDTA, 20 % volume ethanol
8. Elution buffer: 10 millimolar (mM) Tris-HCl (8.0 ), 100 millimolar (mM) NaCl, 1 millimolar (mM) EDTA
9. 3 M sodium acetate (5.2 )
Troubleshooting
Safety warnings
Please read SDS associated with various consumables and kits used in this protocol and wear appropriate PPE. A site specific procedural risk assessment should be carried out prior to introducing this protocol to the lab.
Before start
This protocol is based on the previous description with some modifications [2, 6]. Compared to phenol/chloroform extraction of dsRNA, this method is free of phenol and chloroform and is highly efficient and rapid (less than 2 h to complete whole steps and only requires about 200 mg of fresh material).
This protocol assumes that you have freeze-dried, milled plant tissue material ready.
Methods
3h 24m
Prepare required buffers - list provided in materials
Methods
3h 24m
Measure 50 mg of freeze dried milled plant sample
Methods
3h 24m
Transfer the tissue powder to a 2 mL centrifuge tube containing 600 µL NAE buffer and add pure ethanol to the final concentration of 30 % volume (~257 μL)
2m
Thaw the tissue powder by vortexing immediately and then shake the mixture for at least 00:20:00 at room temperature on a horizontal shaker (1000 rpm, Room temperature ). Avoid foam during vortex.
25m
Centrifuge 10000 x g, 4°C, 00:20:00 .
20m
Equilibrate 40 mg cellulose in 600 µL 1×DSB buffer in a 1.5 mL centrifuge tube.
Equilibration can increase the binding activity of cellulose.
10m
Collect cellulose by centrifuge and discard the supernatant thoroughly.
5m
Transfer the supernatant of step 5 to the centrifuge tube containing equilibrated cellulose from step 7.
2m
Mix well by vortexing, shake the mixture for 00:30:00 at room temperature on a horizontal shaker (1000 rpm, Room temperature ). The incubation time can be increased.
30m
Centrifuge 5000 x g, Room temperature, 00:01:00 to collect the cellulose.
1m
Remove the supernatant and add 1 mL of 1×DSB buffer.
2m
Mix well with a pipette and then shake gently on a horizontal shaker Undetermined, Room temperature , 00:05:00 .
7m
Repeat steps 10–12 twice.
The washing steps can be increased until the supernatant becomes colorless to eliminate the contamination of cytochrome. We repeated the steps twice, as described above.
20m
Remove the supernatant completely by pipetting.
2m
Additional step: centrifuge 5000 x g, Room temperature, 00:01:00 after removing majority of the supernatant, then remove the rest of it.
2m
Add 200 µL elution buffer to the dried pellet, vortex for 00:05:00 on a horizontal shaker (1000 rpm, Room temperature , 00:05:00 ), and then centrifuge5000 x g, Room temperature, 00:01:00 .
6m
Transfer the supernatant to a new 1.5 µL centrifuge tube kept on ice.
2m
Repeat step 15 once.
6m
Collect the supernatant to the same tube of step 16.
1m
Centrifuge the tube containing dsRNA for 5000 x g, 4°C, 00:01:00 and transfer the supernatant to a new 1.5 mL tube to eliminate any remaining cellulose.
1m
Add 1/10 volume of 3 M sodium acetate (5.2 ) (ca. 40 µL ) and 8/10 volume of isopropanol (ca. 320 µL ) to the supernatant, mix well, and incubate overnight at -20 °C or 1h at -80 °C.
Centrifuge 14000 x g, 4°C, 00:30:00 to collect the dsRNA pellet.
30m
Wash the dsRNA pellet with 70% ethanol twice by centrifugation (1000 rpm, 00:01:00 ).
5m
Discard the supernatant, air dry the pellet in a biosafe cabinet for 00:10:00 (extended to 00:20:00 ) and dissolve the dsRNA pellet in about 200 µL molecular biology graded water.
25m
Protocol references
Luan, Y., Fan, W., Korxeelor, X., & Wu, X. (2024). Isolation of dsRNA from Plants by Cellulose Chromatography. In Double-Stranded RNA: Methods and Protocols (pp. 13-17). New York, NY: Springer US.