ISOSPIN Plant RNA
Manual (Ver. 05)

Code No. 310-08171

NIPPON GENE CO., LTD.

IDescription

The ISOSPIN Plant RNA enables a rapid isolation of highly pure total RNA within 1 hour fromplant tissues.

IIKit components
Component(50 preps)

Note

PT Extraction Buffer (for plant)

30 ml x 1

PT Binding Buffer (for plant)

40 ml x 1

(includes ethanol)*PT Wash1 Buffer

40 ml x 1

(includes ethanol)*PT Wash2 Buffer

40 ml x 1

(includes ethanol)*

DNase I (RNase free)

2,000 units x 1

10 x DNase I Buffer

1 ml x 1ddWater (RNase free)

1 ml x 8Spin Column (a Spin Column and Collection Tube set)
*III50 sets

Keep the buffer bottles tightly closed after use.

Storage conditions

DNase I (RNase free) should be stored at -20°C. The remaining kit components can be
stored at room temperature (15-25°C).

IVPrecautions

・ The kit is intended research use only.

1VProtocol

Material not supplied
・Micropipette・Pipette tips
・Pestle・1.5 mL microcentrifuge tubes
・Microcentrifuge(4°C)

Material, as necessary
・liquid nitrogen・Assist Buffer for ISOSPIN Plant RNA (Code No.315-08501)**

See Optional Protocol on Page 6.

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Standard Protocol
1.

Prepare a fresh or frozen tissues Sample (20-100 mg) into a 1.5 mL
microcentrifuge tube.
Note)・To avoid a degradation of RNA, keep samples cold and work quickly in this Step.
・Ensure tissues samples are homogenized in Step 2 immediately after harvest or are frozen
immediately in liquid nitrogen.

2.

Add 600 µl of PT Extraction Buffer (for plant), and homogenize tissuesamples with a Pestle.
Note) To avoid clogging, mix it well.

3.

Centrifuge for 10 min at 13,000 x g at 4°C. Transfer the supernatant to a new
1.5 mL microcentrifuge tube.
Note)・For samples with high lipid content, transfer the supernatant carefully without disturbing thefloating lipid.
・For samples with a high content of extracellular material, centrifuge the supernatantobtained in Step 3 for 10 min at 13,000 x g at 4°C again. Transfer the supernatant to a newtube without disturbing the pellet.

4.

Add an equal amount of PT Binding Buffer (for plant), and mix well byinverting. Spin down lightly.
Example) Add 550 µl of PT Binding Buffer (for plant) to 550 µl of the supernatant.

5.

Prepare a Spin Column (a Spin Column and Collection Tube set). Apply
600 µl of the Mixture to the Spin Column. Centrifuge for 1 min at 13,000 x g
at 4°C.

6.

Discard the flow-through. Place the Spin Column back into the sameCollection Tube.

7.

Apply the remaining Mixture to the Spin Column, and repeat Step 5 and 6.

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8.

Apply 500 µl of PT Wash1 Buffer to the Spin Column.

9.

Centrifuge for 1 min at 13,000 x g at 4°C.

10.

Discard the flow-through. Place the Spin Column back into the sameCollection Tube.

11.

Prepare 100 µl of a DNase I solution from the following into a new 1.5 mL
microcentrifuge tube.
10 x DNase I Buffer

10 µl

DNase I (RNase free)

30 units

ddWater (RNase free)

up to 100 µl

12. Add 100 µl of the DNase I solution to the Spin Column. Incubate for 15 minat room temperature.

13.

300 µl of PT Wash1 Buffer to the Spin Column.

14.

Centrifuge for 1 min at 13,000 x g at 4°C.

15.

Discard the flow-through. Place the Spin Column back into the same
Collection Tube.

16.

Add 600 µl of PT Wash2 Buffer to the Spin Column.

17.

Centrifuge for 2 min at 13,000 x g at 4°C. Discard the flow-through and theCollection Tube.

18.

Transfer the Spin Column to a new 1.5 mL microcentrifuge tube.

19.

Add 50 µl of ddWater (RNase free) to the center of the Spin Column.
Incubate for 3 min at room temperature.

20.

Centrifuge for 1 min at 13,000 x g at 4°C.

21.

Recover your purified RNA solution in the microcentrifuge tube.
Note) The RNA solution can be used directly or stored at -80°C for long term storage.

4Simplified Standard Protocol
Sample (20-100 mg)
Add 600 µl of PT Extraction Buffer (for plant).
Homogenize tissue samples with a Pestle.
Centrifuge for 10 min at 13,000 x g at 4°C.
Transfer the supernatant to a new 1.5 mL microcentrifuge tube.
Add an equal amount of PT Binding Buffer (for plant).
Mix by inverting. Spin down lightly.
Apply 600 µl of the Mixture to a Spin Column. (Keep the remaining Mixture)
Centrifuge for 1 min at 13,000 x g at 4°C.
Discard the flow-through.
Apply the remaining Mixture to the Spin Column.
Centrifuge for 1 min at 13,000 x g at 4°C.
Discard the flow-through.
Prepare 100 µl of a DNase I solution
Add 500 µl of PT Wash1 Buffer.

・ 10 x DNase I Buffer

10 µlCentrifuge for 1 min at 13,000 x g at 4°C.

・ DNase I (RNase free)

30 units

Discard the flow-through.

・ ddWater (RNase free)

up to 100 µl

Add 100 µl of the DNase I solution.
Incubate for 15 min at room temperature.
Add 300 µl of PT Wash1 Buffer.
Centrifuge for 1 min at 13,000 x g at 4°C.
Discard the flow-through.
Add 600 µl of PT Wash2 Buffer to the Spin Column.
Centrifuge for 2 min at 13,000 x g at 4°C.
Discard the flow-through and the Collection Tube.
Transfer the Spin Column to a new 1.5 mL microcentrifuge tube.
Apply 50 µl of ddWater (RNase free) to the Spin Column.
Incubate for 3 min at room temperature.
Centrifuge for 1 min at 13,000 x g at 4°C.
Recover your purified RNA solution in the microcentrifuge tube
5

Optional Protocol
Assist Buffer for ISOSPIN Plant RNA (Code No.315-08501), an Assist Buffer 1 and 2 set, isrequired besides this kit. This optional protocol using the Assist Buffers is beneficial forsamples that are unable to isolate pure and high yield RNA in the Standard Protocol from.

1.

Prior to use, prepare 600 µl of an Extraction Solution; To 500 µl of PT
Extraction Buffer (for plant), add 60 µl of Assist Buffer 1 and 40 µl ofAssist Buffer 2, and mix.
Note)
・The Extraction Solution must be prepared immediately before use because a mixture ofAssist Buffer 1 and Assist Buffer 2 cannot be stored.

2.

Prepare a fresh or frozen tissues Sample (5-100 mg) into a new 1.5 mL
microcentrifuge tube.
Note)
・It is recommended to increase samples gradually.
・If your samples have a high content of extracellular material, the following prewashing might
be more effective. Grind tissue samples lightly with a Pestle in 300 µl of TE within a minute.
Centrifuge for 10 min at 13,000 x g at 4°C, and discard the supernatant.
・To avoid a degradation of RNA, keep samples cold and work quickly in this Step.
・Ensure tissues samples are homogenized in Step 2 immediately after harvest or are frozen
immediately in liquid nitrogen.

3.

Add 600 µl of the Extraction Solution, and homogenize tissue samples witha Pestle.
Note) To avoid clogging, mix it well.

4.

Centrifuge for 10 min at 13,000 x g at 4°C. Transfer the supernatant to a new
1.5 mL microcentrifuge tube.

5.

Centrifuge the supernatant for 10 min at 13,000 x g at 4°C again. Transfer
the supernatant to a new tube.

6.

Add an equal amount of PT Binding Buffer (for plant), and mix well byinverting. Spin down lightly.
Example) Add 550 µl of PT Binding Buffer (for plant) to 550 µl of the supernatant.

67.

Centrifuge the Mixture for 10 min at 13,000 x g at 4°C. Transfer thesupernatant to a new tube.

8.

Prepare a Spin Column (a Spin Column and Collection Tube set). Apply
600 µl of the supernatant obtained in Step 7 to the Spin Column. Centrifuge
for 1 min at 13,000 x g at 4°C.
Note) If the supernatant has not completely passed through the column after centrifugation,centrifuge again.

9.

Discard the flow-through. Place the Spin Column back into the sameCollection Tube.

10.

Apply the remaining supernatant to the Spin Column, and repeat Step 8 and9.

11.

Apply 500 µl of PT Wash1 Buffer to the Spin Column.

12.

Centrifuge for 1 min at 13,000 x g at 4°C.

13.

Discard the flow-through. Place the Spin Column back into the same
Collection Tube.

14.

Prepare 100 µl of a DNase I solution from the following into a new 1.5 mL
microcentrifuge tube.
10 x DNase I Buffer

10 µl

DNase I (RNase free)

30 units

ddWater (RNase free)

up to 100 µl

15. Add 100 µl of the DNase I solution to the Spin Column. Incubate for 15 minat room temperature.

16.

300 µl of PT Wash1 Buffer to the Spin Column.

17.

Centrifuge for 1 min at 13,000 x g at 4°C.

18.

Discard the flow-through. Place the Spin Column back into the sameCollection Tube.

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19.

Add 600 µl of PT Wash2 Buffer to the Spin Column.

20.

Centrifuge for 2 min at 13,000 x g at 4°C. Discard the flow-through and theCollection Tube.

21.

Transfer the Spin Column to a new 1.5 mL microcentrifuge tube.

22.

Add 50 µl of ddWater (RNase free) to the center of the Spin Column.
Incubate for 3 min at room temperature.

23.

Centrifuge for 1 min at 13,000 x g at 4°C.

24.

Recover your purified RNA solution in the microcentrifuge tube.
Note) The RNA solution can be used directly or stored at -80°C for long term storage.

8Simplified Optional Protocol
Assist Buffer for ISOSPIN Plant RNA (Code No.
Prepare 600 μl of an Extraction Solution315-08501) is required besides this kit.

・ PT Extraction Buffer (for plant) 500 µl
Sample (20-100 mg)
Add 600 μl of the Extraction Solution.

・ Assist Buffer 1060 µl

・ Assist Buffer 2040 µl

Homogenize tissue samples with a Pestle.
Centrifuge for 10 min at 13,000 x g at 4°C.
Transfer the supernatant to a new 1.5 mL microcentrifuge tube.
Centrifuge for 10 min at 13,000 x g at 4°C.
Transfer the supernatant to a new 1.5 mL microcentrifuge tube.
Add an equal amount of PT Binding Buffer (for plant), and mix by inverting.
Centrifuge for 10 min at 13,000 x g at 4°C.
Apply 600 μl of the supernatant to a Spin Column. (Keep the remaining supernatant)Centrifuge for 1 min at 13,000 x g at 4°C.
Discard the flow-through.
Apply the remaining supernatant to the Spin Column.
Centrifuge for 1 min at 13,000 x g at 4°C.
Discard the flow-through.
Add 500 μl of PT Wash1 Buffer.

Prepare 100 µl of a DNase I solution

Centrifuge for 1 min at 13,000 x g at 4°C.

・ 10 x DNase I Buffer

10 µl

Discard the flow-through.

・ DNase I (RNase free)

30 units

Add 100 μl of the DNase I solution.

・ ddWater (RNase free)

up to 100 µl

Incubate for 15 min at room temperature.
Add 300 μl of PT Wash1 Buffer.
Centrifuge for 1 min at 13,000 x g at 4°C.
Discard the flow-through.
Add 600 μl of PT Wash2 Buffer to the Spin Column.
Centrifuge for 2 min at 13,000 x g at 4°C.
Transfer the Spin Column to a new 1.5 mL microcentrifuge tube.
Apply 50 μl of ddWater (RNase free) to the Spin Column.
Incubate for 3 min at room temperature.
Centrifuge for 1 min at 13,000 x g at 4°C.
Recover your purified RNA solution in the microcentrifuge tube.
9VI

Troubleshooting

ProblemCause and countermeasure

Low yield

RNA degradation caused by incomplete homogenization or lysis of samples.
Ensure tissue sample is excised as quickly as possible and homogenizedefficiently.

RNA degradation

Immediately after harvesting tissues samples, homogenize in Extraction
Buffer or frozen in liquid nitrogen.
Store frozen samples at -70°C or below.
Use RNase-free solutions and tubes for dissolving RNA.

DNA

Perform a DNase treatment on recovered purified RNA solution if you havecontamination

not performed Step 11 and Step 12.

Variable

Low purity: Extracted RNA is contaminated with impurities such as

absorbancepolysaccharides. See the following countermeasures against Contamination

valueswith impurities.

Contamination

Reduce the amount of samples for Extraction buffer in Step 1 to lyse

with impurities

efficiently.
Centrifuge the supernatant obtained in Step 3 for 10 min at 13,000 x g at 4°C
again. Transfer the supernatant to a new tube without disturbing the pellet
and the float.

Still have trouble

Try the optional protocol on page 6.

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The information in the descriptions of the products may be changed without prior notification.

NIPPON GENE CO., LTD.
If you have any questions, please contact us by web form.

http://www.nippongene.com/siyaku/ISOSPIN Plamt RNA Manual (Ver. 05) 201508-1706

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