ISOSPIN Cell & Tissue RNA
Manual (Ver. 02)

Code No. 314-08211

NIPPON GENE CO., LTD.

IDescription

The ISOSPIN Cell & Tissue RNA enables a rapid isolation of highly pure total RNA within 1
hour from animal tissues or cultured cells.

II Kit components
Component(50 preps)

Note

PT Extraction Buffer (for tissues)

30 ml x 1

C Extraction Buffer (for cells)

30 ml x 1

PT Binding Buffer (for tissues & cells)

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 1

ddWater (RNase free)

1 ml x 8

Spin 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 bestored at room temperature (15-25°C).

IV Precautions
・ The kit is intended research use only.

1V Protocol
Material not supplied
・Micropipette・Pipette tips
・Pestle・1.5 mL microcentrifuge tubes
・Microcentrifuge**Centrifugation of the protocol can be performed at 4°C or 25°C.

Material, as necessary
・liquid nitrogen2

Cultured Cells RNA Extraction Protocol
1.

Add 600 µl of C Extraction Buffer to Sample (up to 3 x 106 culturedcells), and lyse cells by pipetting.

2.

Vortex more than 30 sec.
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.

4.

Add an equal amount of PT Binding Buffer, and mix well by inverting. Spindown lightly.
Example) Add 550 µl of PT Binding Buffer 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 same
Collection Tube.

7.

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

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 same
Collection 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 µl3

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Cultured Cells RNA Extraction Simplified ProtocolSample (up to 3 x 106 cultured cells) in a 1.5 mL microcentrifuge tube
Add 600 µl of C Extraction Buffer, and lyse cells by pipetting.
Vortex more than 30 sec.
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, and 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Animal Tissues RNA Extraction Protocol
1.

Prepare a fresh or frozen tissues Sample (up to 10 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, and homogenize tissue samples with aPestle. Vortex more than 30 sec.
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, and mix well by inverting. Spindown lightly.
Example) Add 550 µl of PT Binding Buffer 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 gat 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.

6

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.

7Animal Tissues RNA Extraction Simplified Protocol
Sample (up to 10 mg)
Add 600 µl of PT Extraction Buffer, and homogenize tissue samples with a Pestle.
Vortex more than 30 sec.
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, and 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.
Centrifuge for 1 min at 13,000 x g at 4°C.
Discard the flow-through.

・ 10 x DNase I Buffer

10 µl

・ DNase I (RNase free)

30 units

・ 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.

8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.

DNAPerform 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.

9VII

Data

Technical Information
RNA Binding Volume

100 μgColumn Volume

900 μlExpected yields
SampleExpected yields

HeLa cell

15 µg RNA/106 cells

Jurkat cells

10 µg RNA/106 cells

Vero cell

15 µg RNA/106 cells

Mouse brain

1.0 µg RNA/mg tissue

Mouse liver

3.5 µg RNA/mg tissue

Mouse kidney

3.0 µg RNA/mg tissue

Mouse testis

1.5 µg RNA/mg tissue

Absorption spectra of RNA
0.12

Mouse brain
マウス脳0.1

マウス肝臓Mouse liver

Abs0.08

マウス腎臓
Mouse kidney
マウス精巣
Mouse testis

0.06
0.04
0.02
0220

240

260
280
Wavelength (nm)10300320

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/

ISOSPIN Cell & Tissue RNA Manual (Ver. 02) 201508-1704

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