ISOVIRUS II
Code No. 312-09091
Manual Ver. 1

Introductions:
The ISOVIRUS II is designed to purify viral double-stranded RNA (dsRNA) for
animal samples, including cultured cells and mammalian tissues. Cellulose
column chromatography allows selective capture of dsRNA. Purified dsRNA issuitable for use in downstream applications such as RNA sequencing, etc.

Experimental Overview.
StepA:

Preparation

ofcellulose-

Step B: Total RNA extraction frompacked column

animal samples

ColumnFilter Column

HomogenizeCollection TubeRNase A and Proteinase K treatment
DNase I treatment

Packed Swollen Cellulose into Filter Column

Mix Ethanol
Keep Supernatant (RNA and EtOH)

Cellulose

Option B-1: When using RNA extraction kits other than this kit
Option B-2: When repurifying dsRNA solution

Step C: Purification of dsRNA

Mixture of
total RNAand EtOH

Bound dsRNA

Bound dsRNA

ssRNA

Cellulose

ssRNA

ssRNA

Bind dsRNA to cellulose

Wash dsRNA

1

Eluted dsRNA
Elute dsRNA

Safety Data Sheet:
TheSafetyDataSheets(SDSs)

areavailableonourwebsite

atwww.nippongene.com.

Kit Contents:
The materials provided are sufficient for 20 preparations.
Component

Quantity

Storage500 μl x 1-20°C

1,000 units x 1-20°C

10 x DNase I Buffer

1.2 ml x 1-20°C

RNase A30 μl x 1

Room temp.*1

VR Extraction Buffer CT1

10 ml x 1

Room temp.

VR Extraction Buffer 2

2 ml x 1

Room temp.*2

2×STE Buffer

7 ml x 1

Room temp.

Filter Column

20 pieces x 1 bag

Room temp.*3

10 tubes x 2 boxes

Room temp.*4

VR Wash Buffer

40 ml x 2

Room temp.*4

VR Elution Buffer

4 ml x 1

Room temp.

Proteinase K
DNase I (RNase free)

Swollen Cellulose

Reagents required but not supplied: ethanol and nuclease-free water.
*1 For long-term storage, store the RNase A at 4°C or -20°C.
*2 If the VR Extraction Buffer 2 contains a precipitate, warm the buffer in a +65°C
water bath and mix to dissolve the precipitate.
*3 A Filter Column consists of a column pre-inserted into a collection tube.
*4 Swollen Cellulose and VR Wash Buffer contain ethanol. Always keep buffer
bottles tightly closed.

Required Materials Not Included:
Pipettors and pipette tips
Micro-pestles (designed for manual homogenization of tissue samples)
1.5 ml microcentrifuge tubes
Centrifuge tubes for cell culture
Centrifuge for spin down2

Microcentrifuge capable of centrifuging 13,000 x g at 4°C
Heat blocks or water baths set to 37°C and 65°C
Vortex mixer
Ice
Ethanol (95-100 %)Water (RNase free)Liquid nitrogen, if necessaryMethod
Step A: Preparation of cellulose-packed column
For each cellulose-packed column, prepare one Filter Column (a column in acollection tube) and one Swollen Cellulose vial (~800 μl).
1. Resuspend the Swollen Cellulose in its tube by through vortexing. Transfer
the Swollen Cellulose from the vial to the Filter Column directly or by pipetting
using a pipette tip with the tip cut off.
2. Incubate for 1-3 minutes at room temperature (leave the cap open) todrain the buffer from cellulose by gravity. If the caps are closed tight, buffer
may not be drained out.
3. Cap the column and spin down for 3 seconds. Remove the column fromthe collection tube and discard flow-through. Re-insert the column in thesame collection tube.
(Stopping Point) Cellulose-packed columns can be stored at 4°C for at least
one week. Close the cap to avoid drying.
4. Slowly apply 500 μl VR Wash Buffer into the column. Do not disturb thepacked cellulose surface.
5. Incubate for 3-5 minutes at room temperature (leave the cap open) todrain the buffer.

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6. Cap the column and spin down for 3 seconds. Discard flow-through and reinsert the column in the collection tube.
7. Slowly apply 900 μl VR Wash Buffer into the column.
8. Incubate for 3-5 minutes at room temperature (leave the cap open) todrain the buffer.
9. Close the cap to avoid drying. The cellulose-packed columns can be stored
at 4°C for up to one day.

Proceed to Step C. Part 1: Bind dsRNA to cellulose.

Step B: Total RNA extraction from animal samples
Samples should be flash frozen or rapidly processed after harvest to remain
RNA intact. Sample input amount: up to 100 mg tissue, 50 mg tissue (withhigh content), or 107 cultured mammalian cells.
1. Transfer animal sample into a clean 1.5 ml microcentrifuge tube.
[For cultured mammalian cells, cells should be pelleted in a centrifuge tube
and all culture medium removed prior to use of this kit.]
2. Add 500 μl of VR Extraction Buffer CT1 to sample and grind tissue using amicro-pestle. For maximal RNA recovery, use a micro-pestle that fits theconical contour of a 1.5 ml microcentrifuge tube.
[For cells, lyse cells by repeated pipetting. Be sure the cell pellet is completely
resuspended in the VR Extraction Buffer CT1.](Note) Incomplete homogenization will reduce the yield of recovered RNA.
3. Add 1 μl RNase A to the tube, mix by inversion.
4. Add 20 μl Proteinase K to the tube, mix by gentle inversion. Do not vortex.
5. Incubate the tube at 65°C for 15 minutes. During incubation, gently mix
every 5 minutes. Do not vortex.
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6. Add 100 μl VR Extraction Buffer 2 to the tube, mix by inversion.
7. Incubate for 3 minutes at room temperature.
8. Add 60 µl 10 x DNase I Buffer and 30 units DNase I (RNase free). Mix byinversion.
9. Incubate the tube at 37°C for 15 minutes. During incubation, gently mix
every 5 minutes. Do not vortex.
10. Add 35 µl Water (RNase free) and 35 µl 2x STE Buffer.
11. Add 143 µl 95 – 100% ethanol to the tube to obtain a final ethanol
concentration of 16%, close the cap, and mix by inversion.
12. Incubate on ice for 10 minutes.
13. Centrifuge for 5 minutes at 13,000 x g at 4°C to pellet cellular debris.
Cellular debris may float on the liquid surface.
14. Transfer supernatant (~900 μl) to a clean microcentrifuge tube, avoiding
the debris. If the volume of supernatant is less than expected, an additional
centrifugation step of 13,000 x g will be required.
15. Close the cap to avoid drying. The supernatant containing 16% ethanol
can be stored at 4°C for up to 4 hours or at -20°C for up to 16 hours.

Proceed to Step C. Part 1: Bind dsRNA to cellulose.

Step C. Part 1: Bind dsRNA to cellulose
For efficient binding of dsRNA to the cellulose-packed column from Step A,
ensure the supernatant from Step B contains 16% ethanol prior to loading
onto the column.

51. Spin down the cellulose-packed column from Step A for 3 seconds.
Remove the column from the collection tube and discard flow-through. Reinsert the column in the same collection tube.
2. Apply the entire supernatant (~900 μl) from Step B to the column.
3. Incubate for 3-15 minutes at room temperature (leave the cap open) todrain the buffer from cellulose by gravity. Depending on the sample
concentration, the buffer may not be drained out even after 15 minutes. In
that case, proceed to the next spin step.
(Note) If cellulose begins to float by air bubbles, flick the tube, open-andclose the cap of the column a few times, or tap the tube lightly on the desk
to settle the cellulose into the column.
4. Cap the column and spin down for 3-5 seconds. Proceed to the next step
to collect the flow-through.
5. Remove the column from the collection tube and collect the buffer fromthe collection tube by pipetting. Re-insert the column into the same collection
tube and re-apply the collected buffer to the column.
6. Incubate for at least 3 minutes at room temperature (leave the cap open).
7. Cap the column and spin down for 3-5 seconds. Proceed to the next step
to collect the flow-through.
8. Remove the column from the collection tube and collect the buffer fromthe collection tube. Re-insert the column into the same collection tube andre-apply the collected buffer to the column.
9. Incubate for at least 3 minutes at room temperature (leave the cap open).
10. Cap the column and spin down for 3-5 seconds. Discard flow-through andre-insert the column in the collection tube.

Proceed to Step C. Part 2: Wash dsRNA.

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Step C. Part 2: Wash dsRNA
Wash 3 times by adding VR Wash Buffer to remove any residual single-strand
RNA and DNA on the cellulose-packed column.
1. Apply 800 μl VR Wash Buffer into the column from Part 1 of Step C towash the bound dsRNA.
(Note) If cellulose begins to float by air bubbles, flick the tube, open-andclose the cap of the column a few times, or tap the tube lightly on the desk
to settle the cellulose into the column.
2. Incubate for 5 minutes at room temperature (leave the cap open).
3. Cap the column and spin down for 3 seconds. Discard flow-through and reinsert the column in the collection tube.
4. Apply 800 μl VR Wash Buffer into the column.
(Note) If cellulose begins to float, to allow the cellulose to settle, flick thetube and so on.
5. Incubate for 5 minutes at room temperature (leave the cap open).
6. Cap the column and spin down for 3 seconds. Discard flow-through and reinsert the column in the collection tube.
7. Apply 800 μl VR Wash Buffer into the column.
(Note) If cellulose begins to float, to allow the cellulose to settle, flick thetube and so on.
8. Incubate for 5 minutes at room temperature (leave the cap open).
9. Cap the column and spin for 5 seconds. Discard flow-through and thecollection tube.
10. Insert the column into a clean 1.5 ml microcentrifuge tube.

Proceed to Step C. Part 3: Elute dsRNA.

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Step C. Part 3: Elute dsRNATo elute dsRNA, nuclease-free water (not included) with the pH 8.0-8.5 or VRElution Buffer (slightly alkaline solution containing 1 mM EDTA) in this kit
could be used. Do not use acidic water.
1. Apply 50 μl VR Elution Buffer or neutral nuclease-free water to the centerof column from Part 2 of Step C so that the elution buffer spreads
throughout the cellulose.
(Note) To increase the concentration, the amount of elution buffer could be
reduced up to 25 µl.
2. Incubate for 5 minutes at room temperature.
(Note) Whether the cap is open or closed does not affect the results.
3. Spin down for 3 seconds. Proceed to the next step to collect the flowthrough.
4. Remove the column and collect the buffer from the tube by pipetting. Reinsert the column into the same tube and re-apply the collected buffer to thecolumn.
5. Incubate for 3 minutes at room temperature.
6. Spin down for 3 seconds. Proceed to the next step to SAVE the flow-through.
7. Remove the column and collect the buffer from the tube by pipetting.
Transfer the collected buffer into a new separate tube and save as first eluted
dsRNA solution.
(Note) The eluted dsRNA solution can be stored at -20°C.
8. Re-insert the column into the same tube and apply 50 μl VR Elution Buffer
or neutral nuclease-free water to the center of column.
(Note) To increase the concentration, the amount of elution buffer could be
reduced up to 25 µl.
9. Incubate for 5 minutes at room temperature.
810. Spin down for 3 seconds. Proceed to the next step to collect the flowthrough.
11. Remove the column and collect the buffer from the tube by pipetting. Reinsert the column into the same tube and re-apply the collected buffer to thecolumn.
12. Incubate for 3 minutes at room temperature.
13. Spin down for 3 seconds. Proceed to the next step to SAVE the flowthrough.
14. Remove the column and collect the buffer from the tube by pipetting.
Transfer the collected buffer into a new separate tube and save as second
eluted dsRNA solution.
(Note) The eluted dsRNA solution can be stored at -20°C.
15. Combine the first eluted dsRNA solution with the second eluted dsRNAsolution to make a total volume of 100 μl dsRNA solution. Alternatively, saveeach 50 μl of eluted dsRNA solution separately.
(Note) The concentration of second eluted dsRNA solution may be lower thanthat of the first eluted dsRNA solution.
(Optional) The eluted dsRNA can be exchanged solution buffers using
ISOSPIN PCR Product (not included, Code No. 315-08001).

Option B-1: When using RNA extraction kits other than
this kit
Instead of Step B, it is possible to combine option B-1 with a nucleic acidsolution or total RNA solution extracted from animal samples using RNA
extraction kit, such us ISOGEN (Code No. 311-02501), ISOSPIN Cell & Tissue
RNA (Code No. 314-08211) and so on.
1. Transfer ~100 μl nucleic acid solution (does not need to be 100 μl) into aclean 1.5 ml microcentrifuge tube.
92. Add 500 μl of VR Extraction Buffer CT1 and 1 μl RNase A to the tube, mix
by inversion.
3. Incubate for 5 minutes at room temperature.
4. Add 20 μl Proteinase K to the tube, mix by gentle inversion. Do not vortex.
5. Incubate the tube at 37°C for 15 minutes. During incubation, gently mix
every 5 minutes. Do not vortex.
6. Add 100 μl VR Extraction Buffer 2 to the tube, mix by inversion.
7. Add 60 µl 10 x DNase I Buffer and 30 units DNase I (RNase free). Mix byinversion.
8. Incubate the tube at 37°C for 15 minutes. During incubation, gently mix
every 5 minutes. Do not vortex.
9. Add 35 µl Water (RNase free) and 35 µl 2x STE Buffer.
10. Add 143 µl 95 – 100% ethanol to the tube to obtain a final ethanol
concentration of 16%, close the cap, and mix by inversion.
11. Incubate on ice for 10 minutes.
12. Centrifuge for 5 minutes at 13,000 x g at 4°C.
13. Transfer supernatant to a clean microcentrifuge tube.
14. Close the cap to avoid drying.
The supernatant containing 16% ethanol can be stored at 4°C for up to 4hours or at -20°C for up to 16 hours.

Proceed to Step C. Part 1: Bind dsRNA to cellulose.

10Option B-2: When repurifying dsRNA solution
In case of contamination of single-stranded RNA in the eluted dsRNA solution
from Step C, you can start with Option B-2 to repurify it.
1. Transfer the dsRNA solution (does not exceed to 350 μl) into a clean 1.5
ml microcentrifuge tube. Add nuclease-free water to bring the volume to 350μl.
2. For every 350 μl dsRNA solution, add 350 μl of 2x STE Buffer to bring thevolume to 700 μl.
3. Add 133 µl 95 – 100% ethanol to the tube to obtain a final ethanol
concentration of 16%, close the cap, and mix by vortex.
4. Close the cap to avoid drying.
The mixture containing 16% ethanol can be stored at 4°C for up to 4 hours
or at -20°C for up to 16 hours.

Proceed to Step C. Part 1: Bind dsRNA to cellulose.

Troubleshooting:Observation

Potential cause

Suggested action

Large sample input

Reduce amount of starting

amounts reduce lysis

material.

efficiency.
Low dsRNA
yield.

Elution buffer has acidic

Use the VR Elution Buffer in thepH.

kit.

Inappropriate ethanol

For dsRNA to bind to the column,concentration.

a final ethanol concentration of
16% is needed.

11References:
Urayama S, Doi N, Kondo F, Chiba Y, Takaki Y, Hirai M, Minegishi Y, Hagiwara
D, Nunoura T. (2020) Front. Microbiol. 11: 561344

Ryo Okada, Eri Kiyota, Hiromitsu Moriyama, Toshiyuki Fukuhara, TomohideNatsuaki (2015) J Gen Plant Pathol 81: 103-107

Ioannis E. Tzanetakis, Robert R. Martin (2008) J Virol Methods 149: 167-170

Morris, T.J., Dodds, J.A. (1979) Phytopathology 69: 854-858.

Richard M. Franklin (1966) Proc Natl Acad Sci U S A. 55: 1504-1511.

Syun-ichi Urayama, Yoshihiro Takaki, Shinro Nishi, Yukari Yoshida-Takashima,Shigeru Deguchi, Ken Takai, Takuro Nunoura (2018) Mol Ecol Resour 18:
1444-1455

Companion Products:
Item

Code No.

Distilled Water, Deionized, Sterile318-90105

Water, Nuclease free

314-09291

ISOSPIN PCR Product315-08001

DNase I (RNase free)

314-08071

RNase A (100 mg/ml)

318-06391

ISOVIRUS (for plant, fungi)

310-08811

ISOGEN

311-02501

ISOSPIN Cell & Tissue RNA

314-08211(Note) This product was developed based on the research results of
new academic field research "Neo-Virology."
For Research Use Only. Not for diagnostic procedures.
NIPPON GENE CO., LTD.
Head office: 1-5, Kanda Nishikicho, Chiyoda-ku, Tokyo 101-0054 Japan
Laboratory: 2-7-18, Toiya-machi, Toyama 930-0834 Japan
www.nippongene.com
ISOVIRUS II Manual Ver.1 (nd2105-en2312)
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