Reagent for RNA Extraction

ISOGENⅡ
Manual (Second edition)
Code No. 311-07361
Code No. 317-07363

NIPPON GENE CO., LTD.

Table of contents

I

Product description

1

II

Product content

1

IIIStorage

1

IVPrecautions

2

V

Protocol2

A: Isolation of total RNA（high molecular weight RNA and small RNA are separately isolated）

3

A-1: Isolation of high molecular weight RNA

3

A-2: Isolation of small RNA

4

B: Isolation of total RNA（high molecular weight RNA and small RNA are isolated in combination） 5
VI

Troubleshooting

VII References

99

ISOGEN II Manual(ver.2)
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I

Product description

ISOGENⅡis a reagent for extracting total RNA and small RNA from animal tissues andcultured cells.
This product is a homogeneous liquid containing phenol andguanidine capable of isolating RNA in a single step byinteracting with cellular components. Unlike the reagents forconventional methods (ISOGEN and ISOGEN-LS), this product

Supernatantdoes not require a liquid phase separation using chloroform.
When ISOGEN Ⅱ is added to a sample to dissolve or
homogenize the sample, and then water is added, DNA, protein

Precipitatesand polysaccharide are precipitated (insolubilized) and can beremoved by centrifugation (right).

High purity RNA can beisolated from the supernatant by ethanol precipitation, and washing and dissolving theprecipitates.
■ Features
・ Chloroform is not used for RNA isolation.
・ Small RNA can be more efficiently extracted compared to using conventional reagents(ISOGEN and the like).
・ High molecular weight RNA (> 200 base) and small RNA (< 200 base) can be fractionated(not fractionating is another method)・ DNA contamination is minimal, and the extracted RNA can be used for RT-PCR andquantitative RT-PCR as it is.
・ RNA can be extracted in about one hour.

IIProduct content

Code No. 311-07361
・ISOGENⅡ
・ManualIII

100 ml1

Code No. 317-07363
・ISOGENⅡ
・Manual

10 ml
1

Storage

Refrigeration
・ Use completely within 6 months from the day of purchase・ This product is shipped at room temperature. After delivery of the product, store at2-10℃ for problem-free use.
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IV

Precautions• This product is a reagent for research and cannot be used for other objectives, e.g.,
as a drug.

Also, this product should be handled only by persons having basic

knowledge of reagents.
• ISOGENⅡ is deleterious substances not for medical use (phenol formulations), andshould be handled with great care.
• When using, wear appropriate protective gear (gloves, goggles etc.).
• Ventilate the area well so as not to inhale vapors.
• If the product enters eye or adheres to skin, wash with large amounts of water for atleast 15 min and consult a doctor.
• Handle this product in accordance with the descriptions in the manual.
• We are not responsible for problems if this product is not handled in accordance withthe manual.
• The product safety data sheet (MSDS) can be reviewed on our website (URL http://www.nippongene.com/pages/products/msds.html)

VProtocol

・ In addition to this product, obtain RNase free water, ethanol and isopropanol.
・ Separately obtain p-Bromoanisole and Glycogen as necessary.
・ Transparent polypropylene tubes may be used. Before using, confirm that the tubes
are rated for the centrifugal force (12 K×g) and ISOGENⅡ(phenol).
・ In the protocol, a method in which 1 ml of ISOGENⅡ is added is described, and in
this case 2.0 ml tubes are required.

However, the extraction scale may be adjusted

to the volume of the centrifuge tube to be used. For example, 1.5 ml tubes can beused by scaling down the protocol to 0.8 (0.8 ml of the supernatant can be recoveredby centrifuging a mixture of about 80 mg of tissue + 0.8 ml of ISOGENⅡ+ 0.32 ml ofRNase free water).
・ All the manipulation can be performed at room temperature but it is recommended
that centrifuging takes place at 4-28℃.
RNA is isolated by the A or B method depending on the sample and the objective.
A:

isolation of total RNA(high molecular weight RNA and small RNA are separately isolated)

B:

A-1:

isolation of high molecular weight RNA

B-1:

isolation of small RNA

isolation of total RNA(high molecular weight RNA and small RNA are isolated in combination)-2-

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A: isolation of total RNA (high molecular weight RNA and small RNA are separatelyisolated)
A-1: isolation of high molecular weight RNA
Sample＊1）(Tissue: about 100 mg; Cells: a culture dish (10 cm2) or about 107cells; Liquid sample: 0.4 ml）
1 ml ISOGENⅡ
Dissolve or homogenize＊2）
0.4 ml（or 0.4 volume） RNase free waterMix by vigorously shaking for 15 sec
Allow to stand at room temperature for 5-15 min＊3）
Centrifuge（12 K×g, for 15 min）＊4）
Supernatant（1 ml: do not withdraw all the supernatant）＊5）
Option＊6）

5 µl（or 0.5% volume）p-Bromoanisole
Mix by shaking for 15 sec
Allow to stand at room temperature for 3-5 minCentrifuge（12 K×g, for 10 min）
Supernatant

0.4 ml（or 0.4 volume） 75% ethanol Mix by inversion
Allow to stand at room temperature for 10 min
Centrifuge（12 K×g, for 8 min）

Precipitates＊7）

Supernatant＊8）
Use for A-2. isolation of small RNA＊9）0.5 ml 75% ethanol

Centrifuge（4 K-8 K×g, for 1-3 min）

Repeat one more time

Precipitates
RNase free water＊10）
High molecular weight RNA solution＊11）（High molecular weight RNA ＞200 base）-3-ISOGEN II Manual(ver.2)
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A-2: isolation of small RNA
Supernatant obtained after the ethanol precipitation in A-1＊8）
2 µl Glycogen＊12）
Vortex0.8 volume of isopropanolMix by inversion

Allow to stand at 4℃ for 30 min
Centrifuge（12 K×g, for 15 min）
Precipitates＊13）
0.5 ml 70% isopropanol＊9）
Centrifuge（4 K-8 K×g, for 1-3 min）

Repeat one more time.

Precipitates

RNase free water＊10）
small RNA solution＊14）（small RNA 10-200 base）-4-ISOGEN II Manual(ver.2)
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B: isolation of total RNA（high molecular weight RNA and small RNA are isolated incombination）
Sample＊1）(Tissue: about 100 mg; Cells: a culture dish (10 cm2) or about 107cells; Liquid sample: 0.4 ml)
1 ml ISOGENⅡ
Dissolve or homogenize＊2）
0.4 ml（or 0.4 volume） RNase free waterMix by vigorously shaking for 15 sec
Allow to stand at room temperature for 5-15 min＊3）
Centrifuge（12 K×g, for 15 min）＊4）

Supernatant（1 ml: do not withdraw all the supernatant）＊5）

5 µl（or 0.5% volume）p-BromoanisoleOption＊6）

Mix by shaking for 15 sec
Allow to stand at room temperature for 3-5 minCentrifuge（12 K×g, for 10 min）
Supernatant
1 ml（or an equal volume） Isopropanol, Mix by inversion
Allow to stand at room temperature for 10 min
Centrifuge（12 K×g, for 10 min）

Precipitates＊7）
0.5 ml 75% ethanol＊9）
Centrifugation（4 K - 8 K×g, for 1-3 min）

Repeat one more time

Precipitates
RNase free water＊10）
total RNA solution＊15）-5-ISOGEN II Manual(ver.2)
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*1）Tissues: Homogenize up to 100 mg of tissue in 1 ml of ISOGENⅡ using a glass-Teflon
homogenizer or polytron homogenizer.

In the case of tissues containing many foreign

substances (such as the liver and spleen), 50 mg of the tissue should be used per 1 ml ofISOGENⅡ. In the method in the protocol, adding 1 ml of ISOGENⅡ is described, but thescale may be adjusted to the volume of the centrifuge tube to be used. The remaining
homogenate can be stored by freezing. See *2).
Attached cells: Remove medium from the culture dish and add at least 1 ml of ISOGENⅡper
3.5 cm dish (10 cm2) and completely dissolve by pipetting. The amount of ISOGENⅡto beused is based on the area of the culture dish, not on the number of cells.
Suspended cells: After precipitating the cells by centrifuge, remove the medium and add atleast 1 ml of ISOGENⅡ per 107 cells and dissolve cells by pipetting.

Since there ispossibility of RNA degradation in cultured cells due to pre-treatment such as trypsin treatment
and washing, add ISOGENⅡ immediately after the removal of the medium. Also, if theamount of ISOGENⅡ to be used is insufficient to the sample, the isolated RNA may becontaminated by DNA.
Liquid sample: Add 1 ml of ISOGENⅡ up to 0.4 ml of the liquid sample and dissolve.
When the sample of less than 0.4 ml is treated, mix the sample with 1 ml of ISOGENⅡ, andthen add RNase free water to bring the volume of the mixture to 1.4 ml. Thereafter, go
through the process of allowing to stand at room temperature for 5-15 min.
Whole blood: ISOGEN-LS is recommended.
Sample with high lipid content: Centrifuge the homogenate once at 12 K×g for 5 min.
Since lipid aggregates on the top layer, collect the supernatant with a pipette or syringe
passed through the lipid layer and transfer to a new tube. By performing the centrifugation at
4-10°C, the lipid layer is solidified and easy to handle.
*2）It is important to quickly excise the tissue and to homogenize thoroughly in order to keep thepurity and yield of RNA high.
・ Immediately homogenize the excised tissue in ISOGENⅡ or freeze in liquid nitrogen.
・ The most effective homogenization method is to crush for 2-3 min with a polytron
homogenizer set at high speed.

Since the brain sample tends to foam, use aglass-Teflon homogenizer.
・ If a sample with high RNase content is used, use cooled ISOGENⅡ to prevent RNA
degradation.
・ To measure tissue, put 1-5 ml of ISOGENⅡ in a tube beforehand and place in anelectronic balance to adjust to zero. Put the fresh tissue immediately after excision or thefrozen tissue in the tube, measure the weight and homogenize immediately.
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crushing the tissue, add the deficit amount of ISOGENⅡ and re-suspend (for example,adjust the ratio to 80 mg of tissue per 1 ml of ISOGENⅡ).
・ The homogenate can be stored at 4°C overnight and at -20°C or -70°C for at least 1 year.
The frozen homogenate can be thawed by incubating at 37-40°C for 5 min.
*3） When 100 mg of tissue, i.e., the maximum amount of the sample that can be processed, istreated with 1 ml of ISOGENⅡ or the sample contains a large amount of DNA, allow tostand for 15 min.
*4） After centrifugation, much of the DNA, proteins, polysaccharides and the like will form blue,
semisolid precipitates at the bottom of the tube. RNA is solubilized in the supernatant.
For example, when 100 mg of tissue is treated with 1 ml of ISOGENⅡ, the volume of theprecipitates of DNA and protein is about 10% of the total volume of the mixture of thehomogenate and water (if 80 mg of tissue is treated with 1 ml of ISOGENⅡ, the ratio would
be about 8%).
*5） Carefully collect 1 ml of the supernatant from the top (75% of the total volume of thesupernatant) and transfer to a new tube. At this time the blue color still remains in thesupernatant. Since the precipitates contain DNA, be careful not to collect the supernatantnear the precipitates in order to prevent DNA contamination.
*6） Since contaminating DNA, proteins, polysaccharides and the like are precipitated byperforming this optional manipulation, this method is effective for samples with high impuritycontent (especially tissue from the liver, kidney, spleen and muscles).
*7） The white precipitates of RNA are attached to the bottom of the tube.
*8） Transfer the supernatant to a new tube and store at 4°C or -20°C to isolate small RNA. At
this time, carefully collect 85% of the supernatant from the top so that the contamination byhigh molecular weight RNA is prevented. The supernatant at this stage retains a pale bluecolor. This supernatant can be stored at -20°C for at least a year.
*9） In this condition, samples can be stored at room temperature overnight, at 4°C for a week
and at -20°C or -70°C for a year.
*10） After removing the supernatant with a micropipette, dissolve the RNA precipitates with anappropriate amount of RNase free water without drying. If the RNA precipitates are dried,
the solubility is markedly reduced.

The RNA precipitates are preferably dissolved byvortexing or pipetting and allowed to stand at room temperature for 2-5 min.

Use

RNase-free tubes.

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containing rRNA and mRNA. These RNA account for about 80-85% of intracellular RNA.
The remaining small RNA fraction can be isolated using protocol A-2.
*12） Small RNA can be efficiently recovered by adding glycogen.
*13） Be aware that the precipitates at this stage are sometimes difficult to see.
*14） RNA isolated here is small RNA of 10-200 base.
*15） RNA isolated here is total RNA containing high molecular weight RNA and small RNA.

Estimated RNA yields obtained using protocols A and B are as follows.

SampleTissueProtocol A-1Protocol B

Liver

5-7 µg RNA/mg tissue

6-8 µg RNA/mg tissue

Kidney, Spleen

3-4 µg RNA/mg tissue

3-4 µg RNA/mg tissue

Skeletal muscles,

0.5-1.5 µg RNA/mg tissue0.5-1.5 µg RNA/mg tissue

1-3 µg RNA/mg tissue

1-3 µg RNA/mg tissue

Brain, Lung
Placenta

6Cultured

Epithelial cells

5-8 µg RNA/10 cells

5-10 µg RNA/106 cells

cells

Fibroblasts

3-5 µg RNA/106 cells

4-6 µg RNA/106 cells

A260/A280 of isolated RNA is 1.7-2.1. Use a buffer of pH 8.0 or higher such as TE (pH 8.0) toaccurately measure absorbance.

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VI Troubleshooting
TroubleCountermeasure

Low yield

Thoroughly homogenize and dissolve the sample. See *2).
Thoroughly dissolve the obtained RNA precipitates.

A260/A280＜1.6

Increase the amount of ISOGENⅡ to be added to the sample.
Use a buffer of pH 8.0 or higher such as TE (pH 8.0) to measure theabsorbance.
Thoroughly dissolve the obtained RNA precipitates.
Since there is a possibility of the contamination of proteoglycan andpolysaccharide, take the countermeasures below.

RNA degradation

Perform ISOGENⅡ treatment immediately after excising the tissue, or
quickly freeze with liquid nitrogen.
Store the frozen samples at -70°C.
For cultured cells, do not perform pretreatments such as trypsin treatment
or washing.
Solutions and tubes used for dissolving RNA should be RNase free.
Perform optional manipulations. See *6).

Contamination

byDNA

Increase the amount of ISOGENⅡ to be added to the sample.
Do not use samples containing organic solvents, strong buffers, salts oralkaline solutions.
After adding water to the homogenate, allow the homogenate to stand for15 min and centrifuge at 16 K×g to precipitate DNA.
Cautiously collect the supernatant after the DNA precipitation. See *5).

Contamination

byRemove fat and precipitates (insoluble materials) by centrifuging (12 K×g,fat, proteoglycan,for 10 min) the homogenate.

and polysaccharide

Perform optional manipulations. See *6).

VII References
1. Chomczynski, P.: Reagents and methods for isolation of purified RNA, US andInternational Patents Pending.
2. Chomczynski, P. and Sacchi, N.: ”Single step method of RNA isolation by acid guanidinium
thiocyanate-phenol- chloroform extraction”, Anal. Biochem., 162, 156-159（1987）
3. Chomczynski, P.: ”A reagent for the single-step simultaneous isolation of RNA, DNA andproteins from cell and tissue samples”, Biotechniques, 15, 532-537（1993）
4. Wilfinger, W., Mackey, K. and Chomczynski, P.: ”Effect of pH and ionic strength on thespectrophotometric assessment of nucleic acid purity”, Biotechniques, 22, 474-481（1997）-9-ISOGEN II Manual(ver.2)
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NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA
930-0834 JAPANTel+81-76-451-6548
URL www.nippongene.com

・The information in the descriptions, specifications and the prices of the products may be changed without priornotification.
・Licensed by Molecular Research Center, Inc. (patent pending)
ISOGEN II Manual(ver.2)
111214KH