Reagent for RNA Extraction

ISOGEN with Spin Column
Manual (4th Edition)

Code No. 318-07511
Code No. 314-07513

NIPPON GENE CO., LTD.

Table of Content

I Product description

2

II Product content

2

III Storage

2

IV Precautions

3

V

3

Protocol<Required reagents, instruments and the like not included in this kit>

3

<Reagents, instruments and the like to be prepared, as necessary>3<Handling procedures>

4<Simplified operation flow>

7

VI Troubleshooting

9

VII Data

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1

I

Product description

The ISOGEN with Spin Column enables a rapid isolation of highly pure total RNA within 1 hour by
utilizing the combination of ISOGEN and a spin column containing silica-gel membranes. Efficient
extraction of total RNA from a variety of samples can be achieved with the use of ISOGEN in asingle-step liquid-phase separation. Also, our unique spin column allows purification of high-quality
RNA in the presence of chaotropic ions using the Boom method.
■ Features• Total RNA can be extracted with high purity.
• No alcohol precipitation is necessary.
• High-quality RNA can be purified from plant samples with high impurity contents.

II Product content
Contents(for 10 extractions)(for 50 extractions)

ISOGEN

10 ml

50 ml

IsoWash I

9 ml

30 ml

IsoWash II

7 ml

30 ml

TE (pH8.0)

1 ml

5 ml

Spin Column

10 sets

50 sets

III

Storage

Store at +2°C to +10°C• Use up this product as soon as possible after opening (within 6 months from the purchase date).
• This product is shipped at room temperature. Upon receipt, store the ISOGEN at 2 to 10°C in thedark. The remaining kit components can be stored at room temperature.

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IV Precautions• This product is intended for research use and not for other purposes. Also, this product should be
handled only by persons having a basic knowledge about reagents.
• ISOGEN contains phenol (a “non-medical deleterious substance”) and should be handled withgreat care.
• Wear appropriate protective gear when handling (gloves, goggles, etc.).
• Avoid inhaling vapor. Use in well ventilated area.
• If the product enters the eyes or adheres to the skin, wash with large amounts of water for at least
15 min and consult a doctor.
• Handle this product in accordance with the descriptions in this manual.
• We are not responsible for problems caused by failure to follow the instructions in this manual.
• For more information, refer to the Safety Data Sheet (SDS) available on our website atwww.nippongene.com

V Protocol<Required reagents, instruments and the like not included in this kit>
• Sample (fresh tissue, frozen tissue, culture cell, plant, etc.)• chloroform• 70% ethanol• Isopropanol• Micropipette• Pipette tips
• 1.5 ml microtubes• Centrifuge (4°C)<Reagents, instruments and the like to be prepared, as necessary>• RNase-free water• Liquid nitrogen• Polytron homogenizer or glass-Teflon homogenizer• Water bath (50°C)
We recommend the use of clear polypropylene tubes for homogenizing sample with ISOGEN.

Make

sure that the tubes can withstand centrifugation at 12,000 x g with ISOGEN (phenol) and chloroform.

3

< Handling procedures >1.

Homogenize sample in 1 ml of ISOGEN (25-100 mg of tissue sample per 1 ml of ISOGEN).
Note) To maximize yield and purity of total RNA, efficient homogenization of the starting material
is a significant requirement.

• Ensure tissue samples are homogenized in ISOGEN immediately after harvest or are frozen
immediately in liquid nitrogen.
• Homogenization of tissue samples can be carried out efficiently using a Polytron
homogenizer at high speed for 2-3 min.
• Before weighing tissue samples, pre-weigh a tube filled with an adequate volume (1-5 ml)
of ISOGEN and set the zero point of balance. As soon as the freshly harvested or frozen
sample is placed in the pre-weighed tube, weigh the total mass to determine the weight of
the tissue. Immediately disrupt and homogenize the sample. Adjust to the appropriate
volume by adding ISOGEN and resuspend the homogenate.
• The homogenate can be stored overnight at 4°C or at least one year at -20°C or -70°C.
When thawing the homogenate, incubate for 5 min at 37-40°C.
Animal tissues: homogenize tissue samples with a glass-Teflon homogenizer or Polytron
homogenizer using up to 100 mg tissue per 1 ml of ISOGEN. When homogenizing impurity-richtissue samples (such as liver and spleen), use 25-50 mg tissue per 1 ml of ISOGEN.
For samples with high lipid content, centrifuge the homogenate at 12K x g for 5 min at 4°C toremove lipid material. Transfer the supernatant carefully without disturbing the floating lipid layer.
Cultured cells: add ISOGEN immediately after removing culture medium. Pretreatment before theaddition of ISOGEN, such as with trypsin or washing cells, is not recommended as it may contribute
to RNA degradation.
A: Adherent cells: remove culture medium from the culture dish, add at least 1 ml of ISOGEN per
3.5 cm dish or 6-well plate (10 cm2), and lyse cells completely by pipetting.

Use the amount of

ISOGEN based on the area of the culture dish and not on cell number.
B: Suspension cells: Collect cells by centrifuging first and remove supernatant (culture medium).
Then add at least 1 ml of ISOGEN per 1 x 107 cells and lyse cells by pipetting (if the number of cells
is unknown, add 10 times volume of ISOGEN to the pellet).
Plant tissues: use up to 100 mg tissue in 1ml of ISOGEN. When homogenizing impurity-richsamples, use 25 mg of sample per 1 ml of ISOGEN.

Freeze sample in liquid nitrogen and pulverize. Homogenize the sample in ISOGEN preheatedat 50°C and incubate for 10 min in a water bath. After incubation, shake vigorously andcentrifuge at 12K x g for 10 min at 4°C. Transfer 1 ml of the supernatant to a new tube carefully
without disturbing the pellet.

When processing samples with high impurity content such as seed and bulb, freeze the resulting
4supernatant at -80°C for 1 hour and thaw by letting stand at room temperature. Centrifuge andtransfer 1 ml of the supernatant to a new tube carefully without disturbing the pellet.
2.

After incubating the homogenate from Step 1 for 5 min at room temperature, add 0.2 times
volume of chloroform per ISOGEN used (200 μl of chloroform per 1 ml of ISOGEN).
Shake the mixture vigorously for 15 sec and store for 2 min at room temperature. Centrifuge
the sample at 12K x g for 15 min at 4°C.
Note)•

Do not use chloroform that contains isoamyl alcohol (such as CIA 24:1).

•

The mixture separates into a lower organic phase, an interphase, and a colorless upper aqueous
phase. RNA remains exclusively in the upper aqueous phase while most of DNA and proteinsremain in the interphase or organic phase.

• The volume of the aqueous phase is about 60% of the volume of ISOGEN used in Step 1. In casethe volume of the aqueous phase is extremely low or is below 30% of the volume of ISOGEN
used, shake the mixture vigorously and re-centrifuge. If this still does not resolve the issue, it isrecommend to repeat procedure with smaller amount of starting material.

3.

Transfer 400 μl of the colorless upper aqueous phase to a new tube.
Note) Transfer carefully without disturbing the interphase and organic phase.

4.

Add an equal amount (400 μl) of 70% ethanol to the aqueous solution from Step 3, and mix byinverting.

5.

Load the entire volume of the mixture from Step 4 (800 μl) onto a Spin Column. Close the lid, andcentrifuge at 12K x g for 15 sec at 4°C.

< Spin Column-based Purification >Apply to the Spin Column

Reset

Spin Column

Collection tube

6.

Centrifuge

Filtrate

Transfer 700 μl of the filtrate to a new microtube and mix with 200 μl of isopropanol. This
mixture will be used in Step 8.
Place the Spin Column back into the collection tube and proceed to Step 7.
Note)• In this step RNA binds to the membrane in the Spin Column; all of the RNA may not binddepending on the presence of impurities derived from the starting materials.
• Do not transfer the entire volume of filtrate. Carefully collect from the top surface without
aspirating impurities left in the bottom (sometimes not visible).

5• Optional DNase treatment may be performed on the Spin Column after performing this step (Seetroubleshooting/options in p.9.)

7.

Apply 500 μl of IsoWash I to the Spin Column from Step 6. Centrifuge at 12 K x g for 15 sec at
4°C. Discard the filtrate.

8.

Apply the entire volume of the mixture from Step 6 (filtrate + isopropanol) to the Spin Column
from Step 7. Centrifuge at 12 K x g for 15 sec at 4°C. Discard the filtrate.
Note) RNA that passed through into the filtrate in Step 6 is bound to the membrane during thisstep. If large precipitates are seen on the membrane, repeat the wash with 300 μl of IsoWash I,
centrifuge at 12K x g for 15 sec at 4°C , and discard the filtrate.

9.

Apply 300 μl of IsoWash II to the Spin Column from Step 8. Centrifuge at 12 K x g for 15 sec at
4°C.

10.

Apply 300 μl of IsoWash II to the Spin Column from Step 9. Centrifuge at 12 K x g for 1 min at
4°C. Discard the collection tube containing the filtrate.

11.

Place the Spin Column from Step 10 into a new 1.5 ml microtube. Apply 20-50 μl of TE or
RNase-free water and leave standing for 1 min.

12.

Centrifuge at12 K x g for 1 min at 4°C at room temperature to elute the RNA.

The table below lists typical yields of RNA from various materials.

SampleExpected Yield

Animal tissues

Brain

0.5 µg RNA/mg tissue(Mouse)Liver

3 µg RNA/mg tissue

Kidney2 µg RNA/mg tissue

Testis

2 µg RNA/mg tissue

Jurkat cells

4 µg RNA/1 x 106 cells

HeLa cells

4 µg RNA/1 x 106 cells

Cabbage sprout

0.3 µg RNA/mg tissue

Rice leaf

0.3 µg RNA/mg tissue

Soybean seed

0.5 µg RNA/mg tissueCorn seed

0.5 µg RNA/mg tissue

Cabbage seed

0.3 µg RNA/mg tissue

Tulip bulb

0.2 µg RNA/mg tissue

Unhulled rice seed

0.2 µg RNA/mg tissue

Cultured cells
Plant tissues

Dilute in a buffer with pH >8.0 to measure absorbance accurately (such as TE, pH8.0).

6<Simplified operation flow>
Add ISOGEN to sample to lyse cells

Add chloroform to homogenate (sample + ISOGEN) and centrifuge
Transfer upper aqueous phase containing RNA to a new tube
Add 70% ethanol to aqueous phase and mix
Apply mixture to Spin Column and centrifuge(Some RNA passes through into

(RNA binds to membrane infiltrate)

Spin Column)
Replace Spin Column into thesameTransfer filtrate to a new tube

collection

tube

after

to

Spin

transferring filtrate

Apply
Add isopropanol to filtrate and mix

IsoWashI

Column. Centrifuge and discard
filtrate. (impurities are washed
away)

Replace Spin Column and apply isopropanol mixture.
Centrifuge. (Some RNA binds in this step)

Continue to the next page

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Continued from the previous page.

Discard filtrate(If large precipitates are seen on the membrane, repeat thewash with IsoWash I)

Apply IsoWash II and centrifuge (15 sec)

Apply IsoWash II and centrifuge (1 min)Discard filtrate and collection tube.

Replace Spin Column onto a new 1.5 ml microtube

Apply TE or RNase-free water and centrifuge

Elute RNA
.

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VI

Troubleshooting

ProblemCause and countermeasure

Low yield

RNA degradation caused by incomplete homogenization or lysis of samples
occurred in cells that are not in contact with ISOGEN solution. Ensure tissue
sample is excised as quickly as possible and homogenized efficiently.
See note in Step 1 in p.4.

RNA degradation

Immediately after harvesting tissue samples, homogenize with ISOGEN or freeze inliquid nitrogen.
Store frozen samples at -70°C or below.
Use RNase-free solutions and tubes for dissolving RNA.

DNA contamination

Increase the amount of ISOGEN to be added to sample.
Do not use samples containing an organic solvent, strong buffer, salt or alkaline
solution.
Do not collect the entire aqueous phase after phase separation. Leave a small
volume of the aqueous phase right above the interphase to avoid aspirating DNA
from the lower phase.
Perform DNase treatment. See options below.

Variable absorbance

Measurement of absorbance values in water may result in variable and inaccurate

valuesvalues.

Measure in a buffer with pH 8.0 or higher, such as TE (pH 8.0), forreproducible results.
Low purity: Extracted RNA is contaminated with impurities such aspolysaccharides.
See the following countermeasures against Low purity and Contamination with
impurities.
Low purity

Insufficient amount of ISOGEN causes insufficient lysis of samples.

Repeatprocedure with half the amount of starting material or with twice volume of
ISOGEN.
Contamination with lipids, proteoglycans, polysaccharides, etc.

See the following

countermeasures against Contamination with impurities.
Contamination with

Remove lipids and insoluble materials by centrifuging the homogenate (12 K x g,

impurities

10 min).

For samples with high impurity content such as seed and bulb, freeze thehomogenate and then centrifuge to remove the impurities.
<Options>
・ DNase treatment for RT-PCR applications: Perform DNase treatment of RNA samples with
Deoxyribonuclease (RT Grade) for Heat Stop (Code No. 312-05951).
・DNase treatment on Spin Column: This can be performed right after Step 6 (before the wash with
IsoWash I in Step 7) with DNase I (RNase free) (Code No. 314-08071). Prepare the DNase solution9

beforehand as below, apply 100 μl of the DNase solution to the Spin Column, and leave standing for 15
min at room temperature. Thereafter, proceed to Step 7.
DNase solution (not supplied and prepare before use): Dilute 30 units of DNase I (RNase free) in 10 μl of
the Reaction Buffer (10 x Buffer) supplied with the enzyme in a tube. Bring up to 100 μl with RNase-freewater and mix by gently pipetting.

VII Data
In accordance with the protocol in this manual, RNA was extracted from mouse liver (50 mg) and frozen
pulverized plant samples (25 mg each) using this product and the absorption spectra were measured.

In

parallel, RNA was extracted using the conventional method (RNA was purified by isopropanol
precipitation after extracted using ISOGEN).
Animal sample: mouse liver

Plant sample: tulip bulb

Column method

Column method

Conventional method

Conventional method

Further, 0.5 μg of RNA calculated based on the absorbance of respective RNA solutions waselectrophoresed in formaldehyde denatured agarose gel.
M

1

2

M

3

4

5

6

7

8

Lane 1: Mouse liver (Conventional method)
Lane 2: Mouse liver (Column method)
Lane 3: Cabbage(Conventional)
Lane 4: Cabbage(Column)
Lane 5: Tulip bulb (Conventional)
Lane 6: Tulip bulb (Column)
Lane 7: Unhulled rice seed (Conventional)
Lane 8: Unhulled rice seed (Column)
M: RNA Ladder (0.125 - 6.0 kb)
1% gel Agarose S. EtBr staining

The results with animal tissue samples showed RNA with high purity equivalent to that of obtainedusing the conventional method was obtained. (similar results with cultured cell samples were also
obtained).
10In comparison of the two methods using the plant samples, the column method using the ISOGEN
with Spin Column produced RNA of higher purity (seen especially in bulbs and unhulled rice seeds)
without impurities that have absorbance near at 230 nm, such as polysaccharides. This can be
confirmed from the result of electrophoresis.

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

http://www.nippongene.com/

・The information in the descriptions, specifications and the prices of the products may be changed without prior notification.

ISOGEN with Spin Column Manual (4th Edition) 201407-1704

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