Fisheries Science 62(5), 727-730 (1996)

Tissue Preservation and Total DNA Extraction from Fish Stored

at Ambient Temperature Using Buffers Containing

High Concentration of Urea

Takashi Asahida,*1 Takanori Kobayashi,*2 Kenji Saitoh,*3 and Ichiro Nakayama*4*1Tohoku National Fisheries Research Instit

ute, Shinhama, Shiogama, Miyagi 985, Japan*2National Research Institute of Aquaculture

, Nansei, Mie 516-01, Japan*3Tohoku National Fisheries Research Institute Hachinohe Branch

, Same, Hachinohe, Aomori 031, Japan

*4National Research Institute of Aquaculture Tamaki Branch, Tamaki, Mie 519-04, Japan

(Received January 12, 1996)

We have developed a high concentration urea containing buffer (TNES-Urea: 6 or 8 mt urea; 10 mM

Tris-HCl, pH 7.5; 125 mM NaCl; 10 mM EDTA; 1% SDS) for DNA extraction by modifying the cell ly

sis buffer for DNA isolation, and we found this buffer is suitable for long-term preservation of tissue

samples from fish at ambient temperatures and for DNA extraction from fish that are rich in cellular en

donucleases. Tissue samples from the Japanese flounder Paralichthys olivaceus and Atlantic herring

Clupea harengus were preserved for periods ranging from 1 month to 3 years and for the Atlantic her

ring transported from Sweden to Japan in TNES-Urea buffer at ambient temperature (10-36•Ž). The

total DNA for each fish was extracted from the muscle or liver tissue which had been preserved for

periods ranging from 1 month to 3 years. The DNA yield was 0.5-2.6 ƒÊg of total DNA/mg tissue. All

DNA from preserved tissues was suitable for DNA analyses, e.g. Polymerase Chain Reaction (PCR)

technique, Southern blot analysis and Random amplified polymorphic DNA (RAPD) analysis. The

TNES-Urea buffer provides a convenient method of tissue preservation and DNA extraction and offers

an alternative to previous methods which require protocols that are restrictive in some field settings.

Key words: tissue preservation, urea, total DNA extraction, PCR, RAPD analysis, Southern blot

analysis, Paralichthys olivaceus, Clupea harengus

The preservation and transportation of tissue samples

for DNA analysis are normally carried out by freezing on

dry ice. However, dry ice is often difficult to obtain in re

mote locations, tropical regions and undeveloped coun

tries, and is not suitable for transportation over long

periods because it sublimates rapidly. Furthermore, dry

ice is restricted on some forms of public transportation.

Although nucleic acids are extremely stable molecules,

they are easily degraded by cellular endonucleases, e.g.

DNase. Moreover, some fish have powerful cellular en

donucleases which prevent DNA extraction. Inhibition or

denaturation of these enzymes is needed for successful tis

sue preservation and DNA extraction.

Proebstel et al.1) reported a tissue preservation method

using a dimethyl sulfoxide (DMSO) salt solution. They

examined samples that were transported at ambient tem

peratures (10-32•Ž) and stored under normal refrigera

tion conditions (5•Ž) for 25-45 weeks, but they did not

test long-term storage of samples at ambient temperatures.

Moreover, their protocol requires the solution to be

changed prior to DNA extraction. Here, we report a buffer

which we have developed and found suitable for long-term

preservation of tissue samples at ambient temperatures

and for total DNA extraction from fish rich in cellular en

donucleases .

Materials and Methods

Fish SamplesWe used Japanese flounder, Paralichthys olivaceus to ex

amine tissue preservation and some DNA analyses, and Atlantic herring Clupea harengus to examine effects of transportation from Sweden to Japan and DNA analysis.

Tissue Preservation and DNA Extraction

We developed the following TNES-Urea buffer: 6 or 8 m

urea; 10 mM Tris-HCl, pH 7.5; 125 mM NaCl; 10 mM

EDTA; 1% sodium dodecyl sulfate (SDS), by modifying

the cell lysis buffer for isolation of total cellular DNA

from animal tissues.2) 50-100 mg of muscles or liver tissue

was removed from each fish and preserved in 400-600 ƒÊl of

the buffer at ambient temperature (10-36•Ž) for periods

ranging from 1 month to 3 years. During the shipping proc

ess, we kept the tissue samples in parafilm-sealed

microtubes. For tissue digestion, 0.8 mg of Proteinase K

was added and the mixture incubated for 8-16 hours at

37•Ž or for more than 3 days at room temperature (20-25•Ž

). The mixture was extracted with phenol-chloroform

(1:1). After the ether extractions, the DNA was precipitat

ed with 2 volumes of ethanol in 0.3 m NaCl and then

resuspended in TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM

EDTA).

728 Asahida et al.

DNA Analysis

We amplified the D-loop containing region of mitochon

drial DNA (mtDNA) by Polymerase Chain Reaction

(PCR). PCR conditions were performed in volumes of 50

ƒÊl containing 10 mm Tris-HCl, pH 8.3; 50 mm KCl, 1.5

mm MgC12, 0.001% gelatin, 200ƒÊM each of dATP, dCTP,

dGTP and dTTP (Takara), 0.2 ƒÊm each of primer 1 and 2,

2.5 unit of Taq DNA polymerase (Takara), and 10-500 ng

of template DNA. The PCR primers for amplification of

the 2.5 kb segment were modified from L15996 by Vigilant

et al. (5•Œ-TCACCC (C/T) T (A/G) (A/G) CT (A/C)

CCAAAGC-3•Œ)3) for the L-strand and complementary of

L1091 by Kocher et al.4) for the H-strand. The PCR

primers for amplification of the 1.1 kb segment were the

same as above for the L-strand and 5•Œ-TGAGACTAT

TTACTTGCACGC-3•Œ for the H-strand. The H-strand

primer was designed from sequence data of the D-loop

region (Saitoh et al., unpublished). Oligonucleotide

primers were synthesized by cyanoethyl-phosphoramidite

chemistry on a Beckman Oligo 1000 DNA synthesizer. Am

plification was performed in a Perkin Elmer GeneAmp

PCR System 9600 DNA Thermal Cycler programmed for

30 cycles of 30 sec at 94•Ž, 30 sec at 55•Ž and 2 min at 72•Ž,

with 5 min at 72•Ž for the final extension step. The

PCR products were digested by 10-15 units of restriction

endonucleases under the manufacturer's recommended

conditions. The restricted PCR products were analyzed by

electrophoresis in 4% Nusieve 3:1 agarose (Takara) gels

and detected by staining with ethidium bromide (EtBr).

We also analyzed restriction patterns of the entire mtDNA

of Japanese flounder using Southern blot hybridizations.

The total DNA extracts underwent enzyme digestion,

0.8% agarose gel electrophoresis in TAE, transfer onto a

nylon membrane (Hibond-N, Amersham) and hybridiza

tion with appropriate probes labeled with digoxigenin

(Boehringer Mannheim). Japanese flounder mtDNA,

purified by two rounds of CsCl ultracentrifugation,sl was

used as the probe for detection of the entire mtDNA.

Hybridization and immunological detection were

preformed using the supplier's recommendations. Ran

dom amplified polymorphic DNA reactions6) were per

formed with arbitrary primers (primer A, 5•Œ-AG

GTCACTGA-3•Œ; B, 5•Œ-TGGTCACTGA-3•Œ; C, 5•Œ

TCACGATGC-3•Œ).6) Amplification was performed in a

Perkin Elmer Gene Amp PCR system 9600 DNA Thermal

Cycler programmed for 40 cycles of 1 min at 94•Ž, 1 min

at 40•Ž and 2 min at 72•Ž. Amplification products were

analyzed by electrophoresis in 2% agarose gels and detect

ed by staining with EtBr.

Results

The total DNA extracted from Japanese flounder was

obtained from muscle or liver tissue which had been

preserved in the TNES-Urea buffer for periods ranging

from 1 month to 3 years (Fig. 1). Also, the total DNA of

Atlantic herring was obtained from muscle tissue which

had been transported from Sweden to Japan and stored at

ambient temperature (20-36•Ž). The DNA yield was 0.5

2.6ƒÊg of total DNA/mg tissue even although it was ex

tracted from extensively (3 years) preserved tissue.

2.5 kb and 1.1 kb segments of the D-loop containing

Fig. 1. Total DNA of Japanese flounder obtained from TNES-Urea

buffer-preserved tissue.

The DNA in lanes 2-7 was extracted from muscle tissue preserved

for 1 month, 3 month, 6 month, 1 year, 2 years and 3 years, respec

tively. Lane 1 contains Hind ‡V digests of ƒÉ phage DNA as a molecu

lar weight marker.

Fig. 2. PCR products of mtDNA of Japanese flounder.

2.5 kb (lanes 2-4) and 1.1 kb (lanes 5-7) segments of the D-loop

containing region of the mtDNA were amplified. Lane 1 contains Sty

I digests of ă phage DNA as a molecular weight marker.

Fig. 3. Dpn ‡U (lanes 2-11) and Taq ƒ¿ I (lanes 12-21) digestion patterns

of PCR products of mtDNA from Japanese flounder.

1.1 kb segments of D-loop containing region of mtDNA were am

plified and digested with 12 restriction endonucleases. Lanes I and

22 contains 100 base pair ladder DNA molecular weight markers

(Pharmacia Biotech).

Tissue Preservation Technique for DNA Analysis 729

Fig. 4. Hind ‡V digestion patterns of entire mtDNA of Japanese

flounder analyzed by Southern blot hybridization.

Lane 1 and 11 contains Sty I digests of ă phage DNA as a molecu

lar weight marker.

Fig. 5. RAPD patterns of genomic DNA of Japanese flounders (lanes

2-4, primer A) and Atlantic herrings (lanes 5-7, primer B).Lane 1 contains 100 base pair ladder DNA molecular weight mar

ker (Pharmacia Biotech).

region of the mtDNA of Japanese flounder were amplified

by PCR (Fig. 2). Some haplotypes were observed in the

analysis of PCR products by restriction endonucleases

(Fig. 3). All DNA extractions from the TNES-Urea buffer

Preserved tissues yielded template DNA which was active

in PCR. No contaminating DNA was detected in the nega

tive controls for extraction or PCR amplifications.

Figure 4 shows Hind ‡V digestion pattern of entire mtD

NA of Japanese flounder as analyzed by Southern blot

hybridization. We have mapped the cleavage sites of

twelve restriction endonucleases in the mtDNA of

Japanese flounder (data not shown). The total DNA ob

tained from 50-100 mg of preserved tissue was sufficient to

Fig. 6. Total DNA of Japanese flounder.

The DNA in lanes 2-10 was extracted from muscle tissue

preserved in 0 M, 1 mt, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M and 8 M urea con

taining buffer, respectively. Lane I contains Hind ‡V digests of d

phage DNA as a molecular weight marker.

perform 20 Southern blot assays.Figure 5 shows the results of an experiment in which

RAPD analysis was performed on genomic DNA from

preserved tissues of Japanese flounder and Atlantic herring. Polymorphic DNA fragments were observed by RAPD analysis using some primers.

Discussion

Although DNA analysis is an indispensable method for fish biology, it is difficult to apply it to samples from remote locations. We have demonstrated that this tissue preservation technique can be used successfully in fish biology. We found that the TNES-Urea buffer is suitable for transportation and long-term preservation of tissue samples at ambient temperature. This preservation technique offers advantages over previous methods which require protocols that may be restrictive in some field settings.

The ability of this buffer to preserve tissue samples de

pends on the concentration of urea used. More than 4 mole (M) urea containing buffer shows good ability for tissue preservation (Fig. 6). Especially high concentrations of urea (6-8 M) containing buffer appear most suitable for long-term storage at high ambient temperatures (more than 30•Ž). There was no degradation of the total DNA extracted from tissues when preserved in 6-8 M urea containing buffer for up to 3 years at ambient temperatures (1036•Ž). Moreover, the total RNA that was extracted from long-term preserved tissue was also detected by agarose gel electrophoresis with EtBr staining. Castelli et al.7) reported some results of DNA fingerprinting in fish using 8 M urea containing buffer for tissue homogenization. Although urea is a denaturant of DNA, it did not influence the results of some DNA analyses. However, in the case of using the 8 M urea containing buffer, it was difficult to recover the aqueous phase when the mixture was extracted with phenol-chloroform, because the aqueous phase was very viscous. To avoid this problem, the mixture should be incubated with Proteinase K for more than 48 hours at

730 Asahida et al.

37•Ž or more than 7 days at room temperature. It is easier

to recover the aqueous phase when using a 4 to 6 M urea

containing buffer than using 8 M urea containing buffer.

We recommend using 8 M urea containing buffer for preser

vation at high ambient temperatures, and 6 M urea for ordi

nary preservation of tissue samples and for tissue transpor

tation. TNES-Urea buffer is suitable for DNA extraction

from fish that are rich in cellular endonucleases such as

Japanese flounder, because urea is an inhibiter of cellular

endonucleases. Also, this buffer is more suitable than an

ordinary buffer for DNA extraction from tissue, because

urea is an activator of Proteinase K. Furthermore, this

buffer is suitable for DNA extraction from formalin fixed

tissue. Aonuma and colleagues tested the efficacy of a

TNES-Urea buffer as a DNA extraction buffer for forma

lin fixed tissue, and suggested its suitability for DNA ex

traction from formalin fixed tissue (personal communica

tion). Also, Takada et al. are studying the cytochrome b

region of mtDNA of an extinct stickleback using DNA ex

tracted from formalin fixed tissue using TNES-Urea buffer

(personal communication). If a method of DNA extrac

tion from formalin fixed tissue is established, museum

stored material will be applicable for DNA study of extinct

species.

The total DNA extracted from preserved fish tissue in

TNES-Urea buffer provided us with some good DNA anal

ysis results, e.g. PCR-RFLP analysis, Southern blot analy

sis and RAPD analysis. Also, we are testing the efficacy of

TNES-Urea buffer as a tissue preservation and DNA ex

traction buffer for a shellfish and a crustacean, and suggest

that it may be useful in the study of invertebrates and ver

tebrates in general.

The TNES-Urea buffer is the most convenient buffer for

tissue preservation and DNA extraction at the present

time. The TNES-Urea buffer coupled with some tech-

niques of DNA analysis, e.g. PCR techniques, Southern blottings and RAPD analysis offers advantages when working with some specimens that live in remote areas or when the transport of frozen samples is not possible.

Acknowledgments We would like to thank Mr. Jun Aoyama and Dr.

Toru Kitamura for technical assistance, Dr. Keisuke Takada and Mr.

Yoshimasa Aonuma for provided information about DNA extraction

from formalin fixed tissues, and Drs. Yoh Yamashita and Chris Norman

for helpful discussion and critical reading of the manuscript.

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