Purification of viral RNA from fluid biological samples

ISOSPIN Viral RNA
INSTRUCTION MANUAL Version 1

Code No. 310-08931

NIPPON GENE CO., LTD.

IIntroduction

ISOSPIN Viral RNA kit is designed for the rapid preparation of viral RNA from fluid biologicalsamples, such as nasopharyngeal and oropharyngeal swab washes, serum, saliva, andsputum.
With the ISOSPIN Viral RNA method, RNA viruses are lysed quickly by NIRV Extraction
Buffer and Proteinase K. Ethanol is added to the lysate and the lysate and ethanol create
appropriate conditions for binding of RNA to the silica-based membrane of the Spin Column.
Impurities are removed by washing with two kinds of Wash Buffers. The RNA is eluted inRNase-free water.
The Spin Column of the ISOSPIN Viral RNA kit enables a high loading capacity. ISOSPIN
Viral RNA kit allows purification of pure RNA in 30 minutes without phenol/chloroform
extraction or alcohol precipitation.

Advantages
・Designed to purify viral RNA in 30 min・High loading capacity of Spin Column and good operability
・Ideal for sensitive applications such as RT-qPCR

Works for a wide variety of viruses, including:・Coronavirus (SARS-CoV-2)
・Poliovirus
・Hepatitis E virus (HEV)・Severe febrile thrombocytopenia syndrome virus (SFTSV)

1IIKit Components

ISOSPIN Viral RNA (Code No. 310-08931)
ItemAmount (50 preps)

Storage Temperature

Proteinase K

1 ml–20°C

NIRV Extraction Buffer

18 mlRoom temperature

NIRV Wash1 Buffer *30 ml

Room temperature

NIRV Wash2 Buffer *30 mlRoom temperature

1 ml

Room temperature

50

Room temperature

50

Room temperature

ddWater3x

Spin Column †Collection Tube2x* Contains ethanol. Always keep buffer bottles tightly closed.
†Contains columns, plus Collection Tube.

Shipping and Storage
All components of the ISOSPIN Viral RNA kit are shipped at room temperature.
Upon receipt, store Proteinase K at –20°C.

NoteIf you want to premix NIRV Extraction Buffer with Proteinase K, you can store thepremixed NIRV Extraction Buffer containing Proteinase K at –80°C for up to 5 days.
Do not store the premixed NIRV Extraction Buffer containing Proteinase K at –20°C.

2

IIICaution

・ This product is intended for general laboratory use and research use only. Not intendedfor any animal or human therapeutic or diagnostic use.
・ Please observe general laboratory precautions, and follow safety guidelines while using
this kit.
・ NIPPON GENE does not assume any responsibility for damages due to improper
application of our products in other fields of application.
・ Please see the Safety Data Sheets available on our website (www.nippongene.com).

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IVProtocol

The ISOSPIN Viral RNA purification procedure is carried out using Spin Columns andstandard microcentrifuge. All centrifugation steps are performed at 8,000 x g at room
temperature.

Sample types include:
・Sputum (pretreated suputum)
・Nasopharyngeal swab (with transport medium for viruses)
・Oropharyngeal swab washes
・Saliva・Serum・Other cell-free body fluids
・Cell-culture supernatants

Equipment and reagents to be supplied by userReagents・96-100% ethanol
Consumables
・Pipette tips
・1.5 ml microcentrifuge tubes
Equipment
・Manual pipettes
・Vortex mixer
・Benchtop Centrifuge
・Microcentrifuge
・Heat block set to 56°C (For sputum samples etc.)4Protocol: Purification of Viral RNA from 140 µl samples1.

Add 20 µl of Proteinase K into a 1.5 ml tube.
Add 360 µl of NIRV Extraction Buffer to the 1.5 ml tube.
Add 140 µl of the fluid sample to the 1.5 ml tube. Vortex for 15 seconds.

2.

Incubate samples at room temperature for 10 minutes.
(For sputum samples, incubate at 56°C for 10 minutes)

3.

Spin down briefly to collect any drops at the bottom of the tube.

4.

Add 400 µl of ethanol. Vortex for 15 seconds. Spin down briefly.
↓5.

Carefully apply the whole tube contents (up to 920 µl of the lysate from step4) to a Spin Column, cap it and place it in a microcentrifuge.

6.

Centrifuge at 8,000 x g for 15 seconds at room temperature.

7.

Discard the flow-through with Collection Tube. Place the column into a new
Collection Tube.
↓8.

Add 500 µl of NIRV Wash1 Buffer to the column.

9.

Centrifuge at 8,000 x g for 15 seconds at room temperature.

10.

Discard the flow-through with Collection Tube. Place the column into a new
Collection Tube.
↓11.

Add 500 µl of NIRV Wash2 Buffer to the column.

12.

Centrifuge at 8,000 x g for 2 minutes at room temperature.

13.

Discard the flow-through with Collection Tube.
↓

14.

Place the column into a clean 1.5 ml tube.

15.

Add 60 µl of ddWater (RNase free) to the center of the membrane. Incubate
at room temperature for 2 minutes.

16.

Centrifuge at 8,000 x g for 2 minutes at room temperature.

17.

The 1.5 ml tube contains your purified viral RNA.

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The flow chart describes the steps for purification of viral RNA from 140 μl samples.

a 1.5 ml tube
Add 20 μl of Proteinase K
Add 360 μl of NIRV Extraction Buffer
Add 140 μl of the fluid sample
Vortex for 15 sec
Incubate at room temperature for 10 min (For sputum, incubate at 56°C for 10 min)Spin down briefly
Add 400 μl of ethanol
Vortex for 15 sec
Spin down briefly
Transfer the tube contents (up to 920 μl of lysate) to a Spin Column
Centrifuge at 8,000 x g for 15 sec
Discard the flow-through. Place the column into a new Collection Tube
Add 500 μl of NIRV Wash1 Buffer
Centrifuge at 8,000 x g for 15 sec
Discard the flow-through. Place the column into a new Collection Tube
Add 500 μl of NIRV Wash2 BufferCentrifuge at 8,000 x g for 2 min
Discard the flow-through with Collection Tube
Place the column into a clean 1.5 ml tube
Add 60 μl of ddWater (RNase free)
Incubate at room temperature for 2 minCentrifuge at 8,000 x g for 2 min
The 1.5 ml tube contains your purified viral RNA

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Optional Protocol: Purification of Viral RNA from 250 µl samples (Scale-Up)1.

Add 20 µl of Proteinase K into a 1.5 ml tube.
Add 350 µl of NIRV Extraction Buffer to the 1.5 ml tube.
Add 250 µl of the fluid sample to the 1.5 ml tube. Vortex for 15 seconds.

2.

Incubate samples at room temperature for 10 minutes.
(For sputum samples, incubate at 56°C for 10 minutes)

3.

Spin down briefly to collect any drops at the bottom of the tube.

4.

Add 350 µl of ethanol. Vortex for 15 seconds. Spin down briefly.
↓5.

Carefully apply the whole tube contents (up to 970 µl of the lysate fromstep 4) to a Spin Column, cap it and place it in a microcentrifuge.

6.

Centrifuge at 8,000 x g for 15 seconds at room temperature.

7.

Discard the flow-through with Collection Tube. Place the column into a new
Collection Tube.
↓8.

Add 500 µl of NIRV Wash1 Buffer to the column.

9.

Centrifuge at 8,000 x g for 15 seconds at room temperature.

10.

Discard the flow-through with Collection Tube. Place the column into a new
Collection Tube.
↓11.

Add 500 µl of NIRV Wash2 Buffer to the column.

12.

Centrifuge at 8,000 x g for 2 minutes at room temperature.

13.

Discard the flow-through with Collection Tube.
↓14.

Place the column into a clean 1.5 ml tube.

15.

Add 60 µl of ddWater (RNase free) to the center of the membrane. Incubate
at room temperature for 2 minutes.

16.

Centrifuge at 8,000 x g for 2 minutes at room temperature.

17.

The 1.5 ml tube contains your purified viral RNA.
7

The flow chart describes the steps for purification of viral RNA from 250 μl samples.
(Scale-Up)

a 1.5 ml tube
Add 20 μl of Proteinase K
Add 350 μl of NIRV Extraction Buffer
Add 250 μl of the fluid sample
Vortex for 15 sec
Incubate at room temperature for 10 min (For sputum, incubate at 56°C for 10 min)Spin down briefly
Add 350 μl of ethanolVortex for 15 sec
Spin down briefly
Transfer the tube contents (up to 970 μl of lysate) to a Spin Column
Centrifuge at 8,000 x g for 15 sec
Discard the flow-through. Place the column into a new Collection Tube
Add 500 μl of NIRV Wash1 Buffer
Centrifuge at 8,000 x g for 15 sec
Discard the flow-through. Place the column into a new Collection Tube
Add 500 μl of NIRV Wash2 Buffer
Centrifuge at 8,000 x g for 2 min
Discard the flow-through with Collection Tube
Place the column into a clean 1.5 ml tube
Add 60 μl of ddWater (RNase free)Incubate at room temperature for 2 minCentrifuge at 8,000 x g for 2 min
The 1.5 ml tube contains your purified viral RNA8V

Troubleshooting
ProblemCause

Solution・ Always use fresh samples for purification.

Poor quality fluid・ Samples should be processed immediately.

biological samples (OftenIf necessary, add RNase inhibitor to theRNA is degraded bysample and ensure appropriate storage

RNases in the starting

conditions up to the processing.

material)

・ Repeated freezing and thawing should beavoided.

Low RNAyield・ To ensure efficient lysis, it is essential that
Incomplete lysis

the sample is mixed thoroughly withProteinase K and NIRV Extraction Buffer.
・ Do not vortex more than 15 seconds. Be

Fragmentation of RNA

especially careful after incubation.
・ Follow the general guideline for handling of

RNase contamination

RNA.
・ Use RNase-free consumables.

Low concentration of virus

・ Follow the optional protocol on page 7 toin the sample

Scale-Up.
・ If possible, pellet the cells using

GenomicDNA

centrifugation, and use supernatant for
isolation of viral RNA.

Samples containing cells

contamination

・ If DNA-free RNA is required, digest theeluate with RNase-free DNase.

Related products
・ Distilled Water, Deionized, Sterile・ DNase I (RNase free)(Code No. 316-90101, 318-90105)(Code No. 314-08071)

・ RNase Inhibitor(Code No. 315-08121)

・ Collection Tube

(Code No. 319-08141, 315-08143)

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VI

Acknowledgment

This product was developed based on the results of joint research with Professor Yoshitomo
Morinaga and Associate Professor Hideki Tani of Department of Microbiology, Graduate
School of Medicine and Pharmaceutical Sciences, University of Toyama.

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NIPPON GENE CO., LTD.
www.nippongene.com

If you have any questions, please contact us by web form.
The information in the descriptions of the products may be changed without prior notification.

ISOSPIN Viral RNA _ Manual (Ver. 1) 202007YMND1