Reagent for RNA Extraction

ISOGENISOGEN-LS
Manual Ver. 3rd

NIPPON GENE CO., LTD.

Table of Content
I.

Principle

2

II.

RNA isolation

3

III.

DNA isolation

13

IV.

Protein isolation

17

V.

References

19

Precautions:• This product is a reagent for research and cannot be used for other objectives. Also, this productshould be handled only by persons having a basic knowledge about reagents.
• ISOGEN and ISOGEN-LS are deleterious substances not for medical use (phenol formulations),
and should be handled with great care.
• When using, wear appropriate protective gear (gloves, goggles, etc.).
• If the product enters eye or adheres to 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 the manual.
• We are not responsible for problems caused if this product is not handled in accordance with themanual.
• The product safety data sheet (SDS) can be reviewed on our website (URL
http://www.nippongene.com/)1

I.

Principle

ISOGEN and ISOGEN-LS are reagents for RNA extraction from humans, animals, plants andbacteria. The liquid phase separation method is used, and DNA and protein can be isolated from asingle sample. Since RNA, DNA and protein can be isolated through a series of manipulations, it isvery effective for analyzing precious samples, and the simplicity of the manipulations allows a large
number of samples to be handled.
ISOGEN and ISOGEN-LS are homogenous liquids containing phenol and guanidine thiocyanate,with ISOGEN-LS containing more phenol than ISOGEN. ISOGEN is used for the samples oftissues and cultured cells, and ISOGEN-LS is effectively used for samples of large volume such as
blood (> 100 μl when processed in a 1.5 ml plastic tube) and liquid samples that are difficult toconcentrate. ISOGEN or ISOGEN-LS is added to a sample, and after dissolving or homogenizing,
chloroform is added to the mixture and centrifuged. The homogenate is separated into 3 phases: anaqueous phase, an organic phase and an intermediate phase. In the aqueous phase, only RNA ispresent, and DNA and protein are present in the intermediate phase and the lower phase. The
aqueous phase is collected, and RNA is precipitated by adding isopropanol. DNA is precipitatedby adding ethanol to the residual intermediate phase and organic phase, and protein is thenprecipitated by adding isopropanol. In this way, RNA, DNA and protein can be sequentially
isolated.
sampleISOGEN or ISOGEN-LS
homogenize or dissolve
Chloroform

Isopropanol
Aqueous phase
Intermediate phase
Organic phase

contains

precipitatesRNA
contains

EthanolDNAwashingRNA

precipitates

washingDNA

precipitates

washing

ProteinandProteinIsopropanol

Storage:
2°C - 10°C
• Use this product as soon as possible after opening the bottle (within 6 months from the purchasedate).
• Do not use the product if discoloration in the product is observed.
• Although the product is shipped at room temperature, it can be used safely by storing it at
2-10°C after delivery.

2

II.

RNA isolation

When isolating RNA using ISOGEN or ISOGEN-LS, intact RNA can be isolated in about one hour
at a high yield. The RNA thus obtained contains almost no DNA or protein, and can be used as-is
for Northern analysis, dot blot hybridization or the like without DNase and other treatments.

[Protocol 1-1, RNA isolation]
The following manipulations should be performed under a clean environment by wearing gloves not
only to prevent RNase contamination but also for safety. Further, chloroform, isopropanol andethanol should be obtained from other sources. Also, tubes made of clear polypropyrene are
preferable.

sample *1)(for ISOGEN; 50-100mg tissue, 10cm2 of culture plate, 5-10 x 106 cells)(for ISOGEN-LS; 0.25ml liquid sample)

1ml ISOGEN or 0.75ml ISOGEN-LS
lysis or homogenization *2)
store for 5min. at room temperature *3)
0.2ml chloroform *4)
shake vigorously for 15sec.
store for 2-3min. at room temperature
12K x g for 15min. at 4°C *5)*6)interphase and organic phasefor subsequent isolation of DNA and proteins [Protocol 2, 3]

aqueous phase *7)
0.5ml isopropanol (or 0.8 volume isopropanol) *5)
store for 5-10min. at room temperature *8)
12K x g for 10min. at 4°C
precipitate *9)at least 1ml 70% ethanol
vortex *10)
7.5K x g for 5min. at 4°C *11)
precipitate
dry briefly *12)
ddH2O, TE (pH8.0) or 0.5% SDS
dissolve
total RNA solution *7) *13)3*1) When samples are treated in a 1.5 ml plastic tube, use ISOGEN for samples with volumes of
100 μl or less, such as the tissues and culture cells. ISOGEN-LS is effective for samples with greater
volume (over 100 μl) such as the blood. Even if the sample volume is large, ISOGEN may be used
if the volume can be reduced to 100 μl or less by concentration. In the case of samples such as
cultured cells, remove fluid such as mediums as much as possible.
When treating a liquid sample using ISOGEN-LS, if the sample volume is less than 0.25 ml, add
water to bring up the volume to 0.25 ml. Make sure that the volume ratio of ISOGEN-LS to thesample is always 3:1. Liquid samples containing many components such as blood and serum
should be diluted two-fold with water, followed by the addition of ISOGEN-LS. Since heparin
inhibits reverse transcriptase activity, sometimes RT-PCR and PCR do not work properly using total
RNA extracted from blood samples collected using heparin. EDTA does not inhibit reverse
transcriptase activity.
*2) The tissues are homogenized with a glass/Teflon or a Polytron homogenizer. Cells grown
attached to culture plates are dissolved by directly adding 1 ml of ISOGEN per 10 cm2(1 well/6-wellplate) by pipetting. In the case of cell suspension, cells are precipitated by centrifuge and thendissolved by adding 1 ml of ISOGEN per 5-10x106 cells by pipetting. Samples such as muscle,
lipocyte and plant, which contain large amount of protein, lipid, sugar and the like, are centrifuged(12K x g, 10 min, 4°C) once after the homogenization, and the supernatant is used for subsequent
manipulations. Note that fat is located in the top layer.
*3) In this state, the sample can be stored at -70°C for at least one month.
*4) Chloroform mixed with isoamyl alcohol may not be used.
*5) The homogenate is separated by centrifugation to an organic phase in the lower layer, theintermediate layer and an aqueous phase in the top layer. The aqueous phase contains RNA but
almost no DNA or protein. The volume of the aqueous phase is about 60% of the volume of added
ISOGEN. If the volume is less than 60%, the volume of isopropanol to be added in the
manipulations thereafter should be 0.8 times the volume of the aqueous phase. If the volume is lessthan 30%, we recommend extracting again after reducing the amount of tissue.
*6) The intermediate phase and organic phase obtained here are stored at 4°C to isolate DNA andprotein, respectively. However, if the intermediate phase and organic phase contain a large amountof insoluble material (sullage, fibrous material and the like), they are not suitable for isolation of
DNA and protein.
*7) To improve the recovery rate of RNA by reducing contamination by DNA and protein, theaqueous phase is transferred to a new tube before performing the subsequent manipulations so thatthe mixing of the organic phase and intermediate phase into the aqueous phase is minimized. If
contamination of genomic DNA is anticipated, extract again by adding an equal volume ofchloroform to the aqueous phase transferred to the new tube. If the presence of contaminants suchas polysaccharide, proteoglycan and glycogen is anticipated or confirmed in the final RNA
preparation, it would be effective to precipitate only RNA by adding the equivalent of half thevolume of the aqueous phase of a high-salt precipitation solution (Code No. 313-06341) andisopropanol and centrifuging at 10K x g for 15 min. Thereafter, the precipitates are washed with4

70% ethanol.
If the amount of RNA is 10 μg or less, the RNA yield can be improved by adding 3 μl of
Ethachinmate to the homogenate or to the aqueous phase.
*8) In this state, RNA precipitates are normally invisible.
*9) RNA precipitates adhere to the inner wall and the bottom of the tube as gel-like pellets.
*10) In this state, the sample can be stored at least for 1 week at 4°C or for 1 year at -20°C.
*11) If RNA precipitates are about to detach from the inner wall of the tube, centrifugation is
performed at 12K x g after ethanol washing.
*12) RNA precipitates are air dried or vacuum dried for 5-10 min. Do not dry using a vacuum
centrifuge. The solubility is drastically reduced when the precipitates are completely dried. Whenthe solubility of the RNA is reduced, OD260/OD280 drops below 1.6, allowing it to be used as amarker. Thus obtained RNA is dissolved in sterilized distilled water, TE (pH 8.0) or 0.5% SDS bysucking with a pipette tip and is preferably incubated at 55-60°C for 10-15 min.
Distilled waterand 0.5% SDS are made RNase-free by diethylpyrocarbonate (DEPC) treatment beforehand, orcommercially available RNase-free products should be used. If TE (pH 8.0) is to be used, acommercial product of RNase-free grade should be used (because Tris-HCl included in TE isdegraded by the DEPC treatment).
*13) When RNA obtained is electrophoresed in agarose gel, apart from the main bands of about 2
kb and 5 kb ribosomal RNA, low molecular RNA of 0.1-0.3 kb and high molecular RNA of 7-15 kb
can be observed. The OD260/OD280 of the RNA obtained is between 1.6 and 1.8. The
measurement of OD260/OD280 is performed by dissolving in water or a buffer of pH>7.5.

5

[Protocol 1-2: RNA isolation from a minute amount of a sample]
For a minute amount of a sample obtained by biopsy or the like, a slight modification of [Protocol
1-1] may work better.
The following is an example of using a minute amount of a sample (rat pituitary gland or needle
biopsy specimen of a human liver). The manipulations should be performed in a clean environment
by wearing gloves not only to prevent RNase contamination but also for safety. Further,chloroform, isopropanol and ethanol should be obtained from other sources. Also, tubes made ofclear polypropyrene are preferable.

sample *1)(1-5mg tissue)

0.8ml ISOGEN
homogenization or pipetting *2)
store for 5min. at room temperature *3)
0.2ml chloroform *4)
shake vigorously for 30sec. *5)
store for 5min. at 4°C
12K x g for 15min. at 4°C *6)*7)

interphase and organic phasefor subsequent isolation of DNA and proteins [Protocol 2, 3]

aqueous phase *8)
12K x g for 15min. at 4°C *9)
aqueous phase *8)
3 μl Ethachinmate *10)
0.8 volume isopropanol
store for 30min. - over night at 4°C *11)
12K x g for 15min. at 4°C
precipitate *12)
1ml 70% ethanol
vortex *13)
7.5K x g for 5min. at 4°C *14)
precipitate
dry briefly *15)
ddH2O, TE (pH8.0) or 0.5% SDS
dissolve
total RNA solution *16)

Results we obtained indicated that the yield of RNA using 1-5 mg of a rat pituitary gland or a needle
biopsy specimen of a human liver was 4-8 μg/1-5 mg wet weight.

6

*1)

For soft tissues (such as liver), RNA can be extracted from a minute amount of tissue.

*2) Pulverize the tissue using a 1ml glass/Teflon homogenizer or by suction/blow-out using apipette tip (1 ml) containing ISOGEN.
*3) In this state, the sample can be stored at least for 1 month at -70°C.
*4) Chloroform mixed with isoamyl alcohol may not be used.
*5) Shake vigorously by hand.
*6) The homogenate is separated by centrifugation to the organic phase in the lower layer, theintermediate layer and the aqueous phase in the top layer. The aqueous phase contains RNA but
almost no DNA or protein. The volume of the aqueous phase is about 60% of the volume of added
ISOGEN. The volume of isopropanol to be added in the manipulations thereafter should be 0.8
volume of the collected aqueous phase. If the volume of the aqueous phase is less than 30%, werecommend extracting again after reducing the amount of the tissue.
*7) The intermediate phase and organic phase obtained here are stored at 4°C to isolate DNA andprotein, respectively.
*8) The aqueous phase is transferred to a new tube before performing the subsequent
manipulations so that the mixing of the organic phase and intermediate phase into the aqueous phaseis minimized. This is to improve the recovery rate of RNA by reducing the contamination by DNA
and protein.
*9) When the supernatant is collected, the recovery rate of the aqueous phase portion can beincreased by re-centrifuging to separate the contaminating organic phase.
*10) The RNA yield can be increased by adding Ethachinmate to the aqueous phase, which also
allows the precipitation of RNA to be visually confirmed.
*11) Contaminants are increased at -20°C or below, and treatment at 4°C is preferable.
state, RNA precipitates are normally invisible.

In this*12) RNA precipitates adhere to the inner wall and the bottom of the tube as gel-like pellets.
*13) In this state, the sample can be stored at least for 1 week at 4°C or for 1 year at -20°C.
*14) If RNA precipitates are about to detach from the inner wall of the tube, centrifugation is
performed at 12K x g after ethanol washing*15) RNA precipitates are air dried or vacuum dried for 5-10 min. Do not dry using a vacuum
centrifuge. Solubility is drastically reduced when the precipitates are completely dried. Whenthe solubility of the RNA is reduced, OD260/OD280 drops below 1.6, allowing it to be used as amarker. The RNA obtained in this manner is dissolved in sterilized distilled water, TE (pH 8.0) or7

0.5% SDS by sucking with a pipette tip and is preferably incubated at 55-60°C for 10-15 min.
Distilled water and 0.5% SDS are made RNase-free by diethylpyrocarbonate (DEPC) treatment
beforehand, or commercially available RNase-free products should be used. When TE (pH 8.0) is
to be used, a commercial product of RNase-free grade should be used (because Tris-HCl included in
TE is degraded by the DEPC treatment).
*16) When RNA obtained is electrophoresed in agarose gel, apart from the main bands of about 2
kb and 5 kb ribosomal RNA, low molecular RNA of 0.1-0.3 kb and high molecular RNA of 7-15 kb
can be observed. The OD260/OD280 of the RNA obtained is between 1.6 and 1.8. The
measurement of OD260/OD280 is performed by dissolving in water or a buffer of pH>7.5.

8[Protocol 1-3: RNA isolation from plant materials]
The total RNA of a plant sample can be obtained by slightly changing [Protocol 1-1].
The following is an example using a plant sample (Arabidopsis or Tobacco). The manipulations
should be performed under a clean environment by wearing gloves not only to prevent RNase
contamination but also for safety. Further, chloroform, isopropanol and ethanol should be obtained
from other sources. Also, tubes made of clear polypropyrene are preferable.

sample *1)(1-3g fresh weight tissue)

grind in liquid nitrogen with the aid of a mortar and pestle
freezed fine powder *2)
10ml ISOGEN/g fresh weight tissue at 50°C *3)
vortex for 30 sec.
incubate for 10min. *4) at 50°C
store for 5min. at room temperature *5)
0.2ml chloroform *6)/1ml ISOGEN
shake vigorously
store for 2min. at room temperature
10K x g for 15min. at 4°C *7)*8)interphase and organic phasefor subsequent isolation of DNA and proteins [Protocol 2, 3]

aqueous phase *9)
0.5ml 4M LiCl *10)/0.5ml aqueous phasemix
store for 1hr. at -70°C *11)
12K x g for 15min. at 4°C
precipitate *12)
0.4ml ddH2O
dissolve
0.4ml isopropanolmix
store for 30min. at 4°C
12K x g for 15min. at 4°C
precipitate
1ml 70% ethanol
vortex
12K x g for 15min. at 4°C
precipitate
dry briefly *13)9

0.4ml ddH2O
dissolve
40 μl 3M sodium acetate
1ml ethanol
mix
store for 1hr. at -70°C
12K x g for 15min. at 4°C
precipitate
1ml 70% ethanol
vortex
12K x g for 15min. at 4°C
precipitate
dry briefly *13)
0.4ml ddH2O
dissolve
total RNA solution *14)

The results we obtained indicated that the yield of RNA using plant samples is 150-200 μg(OD260/OD280≒1.8) from 0.5-1 g of fresh weight of Arabidopsis or Tobacco.
*1) Use samples that are as fresh as possible.
*2)

Freeze samples with liquid nitrogen and pulverize, and then keep frozen.

*3) Put ISOGEN (10-20 ml/g fresh weight tissue) into a centrifuge tube beforehand, incubate at
50°C, and put the sample from *2) therein.
*4) Incubate for 10 min or until the sample is completely dissolved. Take note that when the
incubation time is too long, there is greater polysaccharide contamination.
*5) In this state the sample can be stored for at least one month at -70°C. If the centrifuge tube
used in *3) cannot withstand high centrifugal force (12K x g), subsequent handling will be madeeasier by distributing 1 ml aliquots of the incubated ISOGEN mixture in which the sample is added,
and then by treating the aliquots with chloroform.
*6) Chloroform mixed with isoamyl alcohol may not be used. The amount of chloroform to be
added is 1/5 of the volume of ISOGEN.
*7) The homogenate is separated by centrifugation to the organic phase in the lower layer, theintermediate layer and the aqueous phase in the top layer. The aqueous phase contains RNA but
almost no DNA or protein. The volume of the aqueous phase is about 60% of the volume of added
ISOGEN.

10*8) The intermediate phase and organic phase obtained here are stored at 4°C overnight or -70°C
for several months to isolate DNA and protein, respectively. However, if the intermediate phaseand organic phase contain a large amount of insoluble material (sullage, fibrous material and thelike), they are not suitable for isolating DNA and protein.
*9) The aqueous phase is transferred to a new tube before performing the subsequent
manipulations so that the mixing of the organic phase and intermediate phase into the aqueous phaseis minimized. This is to improve the recovery rate of RNA by reducing the contamination by DNA
and protein. 500 μl of the aqueous phase is put in a tube.
*10) White cloud is formed when lithium chloride is added to the aqueous phase.
*11) When liquid nitrogen is used, leave standing for 15 min.
*12) Jelly-like colorless clear soft precipitates and white precipitates are formed. Remove thejelly-like precipitates as much as possible.
*13) RNA precipitates are air dried or vacuum dried for 5-10 min. Do not dry using a vacuum
centrifuge. If the precipitates are completely dried, the solubility will be drastically reduced.
When the solubility of the RNA is reduced, OD260/OD280 is below 1.6, allowing it to be used as amarker.
*14) If the obtained total RNA solution will not be used immediately, store at -80°C by adding
1/10 volume of 3M sodium acetate and 2.5 volume of ethanol. At the time of use, centrifuge at
10K x g for 15 min, wash the obtained precipitates with 70% ethanol, dry and dissolve in sterilized
distilled water.
When OD260/OD280 is to be measured, dissolve in water or a buffer of pH>7.5.

Estimated yields are as follows:
Sampletissueminute tissue
plant tissue
cultured cells
blood

Yield
6-10 μg RNA/mg tissue6-10 μg RNA/mg tissue
3-4 μg RNA/mg tissue1-1.5 μg RNA/mg tissue1-1.5 μg RNA/mg tissue
1-4 μg RNA/mg tissue
4-8 μg RNA/1-5mg tissue

liver
spleenkidneyskeletal muscle
brainplacenta
rat pituitary gland or humanliver needle biopsy material
Arabidopsis or Tobaccoepithelial cells
fibroblasts(human or animal)

150-200 μg RNA/0.5-1g fresh weight tissue8-15 μg RNA/106 cells
5-7 μg RNA/106 cells
7-15 μg RNA/ml blood11Troubleshooting:ProblemLow yield

OD260/OD280<1.65

Degradation of RNA

DNA contamination

Counter measure• Dissolve (or homogenize) thoroughly after adding ISOGEN or
ISOGEN-LS.
• Dissolve obtained RNA precipitates thoroughly.
• Increase the amount of ISOGEN or ISOGEN-LS to be added to thesample. In this case, increase the amount of additives in the subsequent
manipulations in proportion to the added ISOGEN or ISOGEN-LS.
• After dissolving (or homogenizing) ISOGEN or ISOGEN-LS,
immediately add chloroform without leaving at room temperature for 5min.
• When collecting the aqueous phase, make sure that the phenol phase does
not mix with the aqueous phase.
• Dissolve the obtained RNA precipitates thoroughly.
• Before adding chloroform to the sample, centrifuge at 12K x g for 5 min
to remove precipitates, and then add chloroform.
• Reproducible values can be obtained by dissolving in water or a buffer of
pH>7.5.
• Use a fresh sample.
• In the case of cells, disperse thoroughly with trypsin digestion.
• Perform thorough heat treatment for the solution and containers beingused.
• In agarose gel electrophoresis, confirm that pH of formaldehyde being
used is neutral (degradation occurs at or below pH 3.5).
• Increase the amount of ISOGEN or ISOGEN-LS to be added to thesample. In this case, increase the amount of additives in the subsequent
manipulations in proportion to ISOGEN or ISOGEN-LS.
• If DNA contamination is found in the obtained RNA solution, add 1 ml of
ISOGEN or 0.75 ml of ISOGEN-LS to this solution (100 μl or less) andperform the treatment again.
• Do not use samples containing organic solvents such as ethanol and
DMSO, strong buffer solutions or alkaline solutions.

12

III.

DNA Isolation

DNA is recovered from the intermediate phase and organic phase obtained by centrifugation after
the homogenization in [Protocol 1-1, 1-2, 1-3]. The isolated DNA can be used as a substrate for
PCR and restriction enzymes.
However, if the intermediate phase and organic phase obtained in [protocol 1-1, 1-2, 1-3] contain alarge amount of insoluble material (sullage, fibrous material and the like), they are not suitable forisolating DNA because the yield may be small and the purity may be low.

[Protocol 2: DNA isolation]
The following manipulations should be performed while wearing gloves for safety. Further,ethanol and sodium citrate should be obtained from other sources. [Protocol 2] corresponds to[Protocol 1-1] when 1 ml of ISOGEN is used.

interphase and organic phase [protocol 1-1] *1)
0.3ml ethanol
mix by inversion
store for 2-3min. at room temperature
2K x g for 5min. at 4°C *5)
supernatantfor subsequent isolation of proteins [Protocol 3]

precipitate

*3)

once

1ml 0.1M sodium citrate in 10% ethanol *2)
store for 30min. at room temperature (with periodic mixing)
2K x g for 5min. at 4°C

precipitate
2ml 70% ethanol
store for 10-20min. at room temperature (with periodic mixing) *4)
2K x g for 5min. at 4°C
precipitate
dry briefly *5)
ddH2O or TE (pH8.0)
dissolve *6)
DNA solution *7)13*1) To obtain high-purity DNA, the remaining aqueous phase must be carefully and completely
removed. The intermediate phase and the organic phase can be kept overnight at 4°C.
*2) [Preparation method of 0.1 M sodium citrate in ethanol]
Add sterilized distilled water to 10 ml of 1M sodium citrate and 10 ml of ethanol to bring up thevolume to 100 ml and mix.
[Preparation method of 1M sodium citrate]
Dissolve 29.4 g of tri-sodium citrate 2 hydrate in water, bring up the volume to 100 ml andsterilize by autoclaving at 121°C for 20 min.
*3) Do not reduce washing time. Wash longer if there is more than 200 μg DNA or very large
amounts of components other than DNA.
*4) The samples in this state can be stored for several months at 4°C.
*5) Dry in vacuum for 5-10 min.

When completely dried, DNA is very difficult to dissolve.

*6) Dissolve by gently sucking with a pipette. At this stage, especially when DNA is isolated
from tissue, the sample may still contain an insoluble gel-like substance. In such cases, the gel-like
substance is removed by centrifugation (12K x g, 10 min). To further dissolve the insoluble
substance, addition of NaOH to a final concentration of 8 mM may be effective. The sample can be
stored overnight at 4°C in this state. For longer storage, the sample can be dissolved in TE (pH 8.0)
and stored as-is or the sample can be dissolved in sterilized distilled water and adjusted to final
concentrations of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. When dissolved in NaOH, pH isadjusted to 7-8 by HEPES by referring the table below, and EDTA is added to a final concentrationof 1 mM.
*7) At this time, insoluble precipitates are found. To further increase the purity, a phenol /chloroform treatment may be performed. However, in this case the yield may be decreased.
When DNA is isolated in accordance with [Protocol 2], 60-100 kb DNA accounts for about 70% andabout 20 kb DNA accounts for about 30% of total DNA isolated from cells, and 60-100 kb DNA
accounts for about 80% and about 20 kb DNA accounts for about 20% of total DNA isolated from
cultured cells. However, since the average length of DNA depends on shearing force duringhomogenization, the use of a strong homogenizer such as a Polytron homogenizer should be avoided
to obtain larger DNA fragments. For the obtained DNA solution, the yield is calculated bymeasuring OD260 after diluting with water. 1 OD260 = 50 μg dsDNA/ml. Rough estimation of theyields for 106 cells of human, rat and mouse are 7.1 μg, 6.5 μg and 5.8 μg, respectively. The DNA
obtained in this manner contains almost no RNA or protein, and OD260/OD280 is 1.7 or higher.
When used as a substrate of PCR and restriction enzymes, DNA dissolved in sterilized distilledwater or TE (pH 8.0) can be used as-is, but for DNA dissolved in NaOH at a final concentration of 8
mM, it is necessary to adjust pH beforehand by HEPES addition or dialysis. The table below may
be referred to for adjusting the pH by HEPES. 80-90% of the DNA obtained by this method can becleaved by 3-5 units of restriction enzyme per 1 μg for 3-24 hours.
OD260/OD280 is measured by dissolving DNA in water or a buffer of pH>7.5.

14

final pH
8.4
8.2
8.0
7.8
7.5
7.2
7.0

required HEPES
concentration (M)
0.1
0.1
0.1
0.1
0.1
1
1

The estimation of the yield is as follows.
sampletissuesliver
kidneyskeletal muscles
brain
placenta
cultured cells (human, rat and mouse)required HEPES volume(μl/ml 8 mM NaOH)
86
93
101
117
159
23
32

yield
3-4 μg DNA/mg tissue
3-4 μg DNA/mg tissue
2-3 μg DNA/mg tissue
2-3 μg DNA/mg tissue
2-3 μg DNA/mg tissue
5-7 μg DNA/106 cells

15Troubleshooting:ProblemLow yield

OD260/OD280<1.70

Degradation of DNA

Contamination by RNA
Formation of Insoluble
precipitates

Counter measure• Thoroughly dissolve (or homogenize) after adding ISOGEN or
ISOGEN-LS.
• Thoroughly dissolve obtained DNA precipitates.
• If insoluble precipitates are found in the obtained DNA solution,
disrupt the insoluble precipitates with a pipette tip or the like, centrifuge
after incubating at 55°C for 20 min, and then use the supernatant.
• Completely remove phenol by repeating the DNA wash with 0.1M
sodium citrate in 10% ethanol. Also, DNA solution loosened by
pipetting is centrifuged and the supernatant can be treated withchloroform. However, in this case the recovery rate may be lower.
• Reproducible values can be obtained by dissolving in water or a bufferof pH>7.5.
• Use a fresh sample.
• Avoid homogenizing samples using a high speed homogenizer such asa Polytron.
• Completely remove the aqueous phase when RNA is isolated.
• Thoroughly wash DNA with 0.1 M sodium citrate in 10% ethanol.
• Insoluble precipitates may be found when extracted DNA is suspended
in sterilized distilled water or TE (pH 8.0). In such cases, centrifuge
and use the supernatant. If the yield of DNA obtained from thesupernatant is low, sodium hydroxide may be added at a final
concentration of 8 mM. Suspending by adding sodium hydroxide may
facilitate the solubilization of the insoluble precipitates. To further
increase purity, a phenol/chloroform treatment can be performed, but
the yield may be reduced.

16

IV.

Protein isolation

Protein is recovered from the organic phase of the supernatant obtained by the centrifugation of theethanol precipitates from [Protocol 2]. The isolated protein can be used as-is for Western blotting.

[Protocol 3: Protein isolation]
The following manipulations should be performed while wearing gloves for safety. Guanidinehydrochloride, SDS, ethanol and isopropanol should be obtained from other sources. [Protocol 3]
corresponds to [Protocol 1-1] when 1 ml of ISOGEN or 0.75 ml of IOSGEN-LS is used.

supernatant [protocol 2] *1)
1.5ml isopropanol
store for 10min. at room temperature
12K x g for 10min. at 4°C
precipitate

twice

1ml 0.3M guanidine hydrochloride in 95% ethanol *2)
store for 20min. at room temperature
7.5K x g for 5min. at 4°C

precipitate
2ml ethanol *2)
store for 20min. at room temperature
7.5K x g for 5min. at 4°C
precipitate
dry briefly *3)
1% SDS
dissolve *4)
protein solution *5)

17*1) In this protocol, the amount of the supernatant is about 0.8 ml.
by dialyzing against 0.1% SDS at 4°C.

The yield may be increased*2) In this state, the sample can be stored at least for 1 month at 4°C or for at least one year at-20°C.
*3) Vacuum dry for 5-10 min.
*4) The following 3 methods can be used for effectively recovering protein.
A) Dissolve pellets in 1% SDS by pipetting and incubate at 50°C. Since the pellets contains
insoluble material, remove them by centrifugation (10K x g, 10 min, at 4°C).
B) Dissolve pellets in 10M urea/50 mM DTT by disrupting with a needle (use a fresh
urea/DTT solution). Store the solution at room temperature for 1 hour and heat in aboiling water bath for 3 min. Cool down in ice and sonicate. If not dissolved, performboiling and sonication twice more until pellets are dissolved.
C) Dialyze the supernatant of phenol/ethanol from *1) against 0.1% SDS at 4°C.
*5) Insoluble materials should be removed by centrifugation (10K x g, 10 min, at 4°C). The
protein thus obtained can be used as-is for Western blotting and can be stored at -20°C.

Troubleshooting:ProblemLow yield

Degradation of protein
Band deformation in PAGE

Counter measure• Thoroughly dissolve (or homogenize) after adding ISOGEN or
ISOGEN-LS.
• Thoroughly dissolve obtained protein precipitates.
• Use a fresh sample.
• Thoroughly wash protein precipitates.

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V.

References

1. Chomczynski P and Sacchi N (1987) Single step method of RNA isolation by acid guanidinium
thiocyanatephenol-chloroform extraction. Anal Biochem,162, 156-159.
2. Chomczynski P (1993) A reagent for the single-step simultaneous isolation of RNA, DNA andproteins from cell and tissue samples. BioTechniques,15, 532-537.
3. Mackey K and Chomczynski P (1996) Long-term stability of RNA isolation reagents. J NIH Res.,8, 72.
4. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A and Struhl K (1990)
Appendix 1, in Current Protocols in Molecular Biology, vol 2, p. A.1.5, John Wiley and Sons,
Inc., New York, NY.
5. Chomczynski P and Mackey K (1995) Modification of the TRI Reagent procedure for isolationof RNA from polysaccharide - and proteoglycan - rich sources. Biotechniques,19, 942-945.
6. Wilfinger W, Mackey K and Chomczynski P (1997) Effect of pH and ionic strength on the
spectrophotometric assessment of nucleic acid purity. BioTechniques, 22, 474-481.
7. Varela L and Margot Ip (1996) A multifunctional regulator of mammary gland development.
Endocrinology. 137(11), 4915-4924.

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http://www.nippongene.com/

ISOGEN, ISOGEN-LS Manual Ver. 3rd 20110921-1803

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