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Forensic Science International: Genetics Supplement Series 5 (2015) e39e41

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Forensic Science International: Genetics Supplement Series


journal homepage: www.elsevier.com/locate/FSIGSS

Effects of storage method on DNA degradation in old bloodstain


samples
M. Haraa,* , H. Nakanishib , S. Takahashic , A. Tamurad , K. Yoneyamaa , K. Saitob , A. Takadaa
a

Department of Forensic Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Saitama 350-0495, Japan
Forensic Medicine, Juntendo University School of Medicine, 2-1-1Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan
Department of Forensic Medicine, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
d
Department of legal Medicine, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka, 569-8686, Japan
b
c

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 20 August 2015
Accepted 7 September 2015
Available online 10 September 2015

The effects of different storage methods on both DNA degradation and the results of preliminary blood
tests were evaluated. Bloodstains stored at room temperature, 4  C, 20  C, and 80  C for 20 years; whole
blood samples stored at 20  C and 80  C for 20 years; and fresh whole blood samples were analyzed.
DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41 bp
fragments. Preliminary testing included leuco-malachite-green staining, anti-human hemoglobin (Hb)
testing by immunochromatography, and the detection of the hemoglobin-beta (HBB) mRNA. Bloodstains
stored at room temperature and 4  C were more highly degraded than fresh blood, as indicated by the
ratio of 129:41 bp DNA fragments and 305:41 bp DNA fragments. All samples tested positive using leucomalachite-green staining and anti-human Hb assays. HBB was not detected in any whole blood samples
stored at 20  C or 80  C, although HBB was detected in all bloodstain samples. Therefore, to prevent
DNA degradation during long-term storage, it is recommended that blood samples be stored at below
20  C. In addition, stored bloodstains are suitable for preliminary tests of blood, rather than storing
blood.
2015 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Storage condition
DNA degradation
Leuco-malachite-green test
Anti-human Hb test
mRNA detection

1. Introduction
When seeking a resolution to a long-unsolved crime, short
tandem repeat (STR) genotyping of old bloodstains is evidentially
important. However, forensic data may be difcult to obtain if
samples have been improperly stored. In the present study, we
explored the effects of different storage conditions on DNA
degradation and preliminary blood tests.
2. Materials and method
2.1. Samples
Bloodstains that adhered to cotton gauze and were stored at
room temperature, 4  C, 20  C, and 80  C for 20 years (n = 6,
each); whole blood samples stored at 20  C and 80  C for
20 years (n = 6, each); and fresh whole blood samples (n = 6) were

* Corresponding author. Fax: +81 49 276 1177.


E-mail address: ms2_hara@saitama-med.ac.jp (M. Hara).
http://dx.doi.org/10.1016/j.fsigss.2015.09.016
1875-1768/ 2015 Elsevier Ireland Ltd. All rights reserved.

analyzed. This study was approved by the Ethics Committee at


Saitama Medical University.
2.2. DNA extraction and assessing DNA degradation
DNA was extracted and puried from a 1 1-cm piece of gauze
or 30 mL of whole blood using the EZ1 DNA Investigator Kit
(QIAGEN, CA, USA), according to the manufacturers instructions,
and was eluted in 50 mL water. DNA degradation was assessed by
calculating the ratios of 129 and 305 bp DNA fragments to 41 bp
DNA fragments using the KAPA Human Genomic DNA Quantication and QC Kit (Kapa Biosystems, Woburn, MA, USA), according to
the manufacturers instructions. The 41-, 129- and 305-bp DNA
fragments were quantied using the StepOne-Plus Real-Time PCR
System.
2.3. Discrimination test for blood
Preliminary testing was performed using the leuco-malachitegreen test, anti-human Hb testing by immunochromatography,
and detection of blood-specic mRNAs. The leuco-malachite-green
reagent was spotted directly onto bloodstains and incubated at

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M. Hara et al. / Forensic Science International: Genetics Supplement Series 5 (2015) e39e41

Fig. 1. The DNA degradation ratio in bloodstains stored at room temperature, 4  C, 20  C, and 80  C for 20 years; of whole blood samples stored at 20  C and 80  C for
20 years; and of fresh whole blood. Mean values  SD for six samples each are shown. Dark gray bars represent the 129:41 bp ratio, whereas the light gray bars represent the
305:41 bp ratio. *Signicant differences were calculated using the t-test (P < 0.05), compared with fresh whole blood.

room temperature. A positive result was taken as the visible


detection of bluegreen coloration after a 1 min incubation.
Anti-human Hb testing by immunochromatography was
performed using OC-Hemocatch S (Eiken Chemical, Tokyo, Japan)
with a 0.2  0.2-cm bloodstain, according to the manufacturers
protocol.
Total RNA was extracted from a 1 1-cm piece of gauze or 30 mL
of whole blood using the RNeasy Mini Kit (QIAGEN). DNase I
digestion and reverse transcription were performed using the
RNase-Free DNase Set (QIAGEN) and High-capacity RNA-tocDNATM Kit (Life Technologies, Carlsbad, CA, USA), respectively,
according to the manufacturers instructions. To investigate the
expression of blood-specic mRNA, the hemoglobin-beta (HBB;
Hs00758889_s1) gene was selected; and the 18S rRNA (18S;
Hs99999901_s1) gene was chosen as an endogenous control. All
primers and probes were supplied in a commercially designed kit
(Life Technologies). Real-time PCR was performed in 20-mL
reaction mixtures containing 2 TaqMan1 Fast Advanced Master
Mix (Life Technologies), a 20 TaqMan1 Gene Expression Assay
reagent (primers and probe; Life Technologies) and 2 mL of cDNA.
PCR amplication was performed using the StepOne-Plus RealTime PCR System (Life Technologies) programmed for 2 min at
50  C and 10 min at 95  C, followed by 40 cycles of 15 s at 95  C and
1 min at 60  C. When the threshold reached 0.2, HBB detection was
dened as a sample that had a Ct value of 34 cycles, whereas 18S
detection was dened as a sample that had a Ct value of 33 cycles.

3. Results and discussion


Fig. 1 shows the relationship between storage conditions and
DNA degradation. Bloodstains stored at room temperature and 4  C
were signicantly more degraded than fresh blood samples, as
determined by the 129:41 bp and 305:41 bp fragment ratios.
Consistent with the mRNA data, sample storage at 20  C or lower
is suitable for maintaining the integrity of DNA. Hara et al. reported
that the Identiler kit did not detect all loci when the 305:41 bp
ratio was 0.0118 [1]. In this study, the 305:41 bp ratio of
bloodstains stored at room temperature for 20 years was
0.0220  0.0130 (mean  standard deviation) (data not shown);
thus, storage under these conditions might affect STR genotyping.
Table 1 shows the number of positive reactions in preliminary
tests examining various storage conditions. All bloodstains and
whole blood samples tested reacted positively with leucomalachite-green staining and anti-human Hb testing. In whole
blood samples stored at 20  C and 80  C, 18S was detected in all
samples, whereas HBB was not detected. In comparison, HBB and
18S were detected in all bloodstain samples. These data suggest
that stored bloodstains are suitable for preliminary tests of blood,
rather than storing blood. In conclusion, to prevent DNA
degradation, blood samples should be stored at below 20  C for
long-term storage. In addition, stored bloodstains are suitable for
preliminary tests of blood, rather than storing blood.

Table 1
The number of positive reaction of preliminary tests in various storage conditions.

Bloodstain RT
Bloodstain 4  C
Bloodstain 20  C
Bloodstain 80  C
Whole blood 20  C
Whole blood 80  C
Fresh whole blood

Leuco-malachite-green staining test

Anti-human Hb test

mRNA detection
HBB

18S

6
6
6
6
6
6
6

6
6
6
6
6
6
6

6
6
6
6
6
6
6

6
6
6
6
0
0
6

6
6
6
6
6
6
6

M. Hara et al. / Forensic Science International: Genetics Supplement Series 5 (2015) e39e41

Conict of interest statement


None.
Role of funding
No funding.

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References
[1] M. Hara, H. Nakanishi, S. Takahashi et al. Relationship between DNA degradation
ratios and the number of loci detectable by STR kits in extremely old seminal
stain samples, Legal Med. In press.

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