Legal Medicine 11 (2009) S43–S45

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Legal Medicine
journal homepage: www.elsevier.com/locate/legalmed

Postmortem quantitative mRNA analyses of death investigation in forensic

pathology: An overview and prospects
Dong Zhao a,*, Takaki Ishikawa a, Li Quan a, Tomomi Michiue a, Bao-Li Zhu a,b, Hitoshi Maeda a
a
Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, 545-8585 Osaka, Japan
b
Department of Forensic Pathology, China Medical University School of Forensic Medicine, No. 92, Beier Road, Heping District, Shenyang, 110001 Liaoning, China

a r t i c l e i n f o a b s t r a c t

Article history: To analyze pathophysiological dynamics of the death process using mRNA quantification, previous stud-
Received 9 December 2008 ies investigated pulmonary surfactant-associated protein (SP-A), as well as hypoxia-inducible factor 1
Accepted 8 January 2009 (HIF-1) and its downstream factors. Quantitative assays of these mRNA transcripts were established
Available online 6 March 2009
using TaqMan real-time RT-PCR. Experimental studies showed that most of these factors in forensic
autopsy materials gradually degraded in patterns similar to those of endogenous references during the
Keywords: early postmortem period within 48 h; postmortem interference might not usually be significant in rela-
Forensic pathology
tive mRNA quantification. Subsequent mRNA analyses of these factors in serial autopsy cases suggested
mRNA quantification
Hypoxia-inducible factor 1
their potential usefulness to investigate the pathophysiology of the death process. Further analyses of
Erythropoietin VEGF and GLUT1 mRNA in the lung and skeletal muscle shed light on tissue ischemia/hypoxia and sub-
Vascular endothelial growth factor sequent tissue-dependent pathological changes leading to death after injury. Animal experiments partly
Glucose transporter supported the above-mentioned findings and also suggested further potential mRNA targets for practical
use. These studies on postmortem quantitative mRNA analyses might offer insight into pathophysiolog-
ical mechanisms in the death process, suggesting that systemic postmortem quantitative mRNA analyses
from multi-faceted aspects of molecular biology can be developed and incorporated into death investiga-
tions in forensic pathology, to support and reinforce morphological evidence.
Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Steady mRNA patterns in cells are controlled by precise up-/

Molecular forensic science has been dominated by DNA re-
search over the past decade, leading to the success of DNA technol-
ogy in criminalistics. However, the reportedly rapid postmortem
and in vitro decay of RNA has kept forensic scientists from investi-
gating this field.
However, the situation is changing with accumulating reports
on the enormous potential of the novel RNA technology of real-
time reverse transcription-polymerase chain reaction (RT-PCR)
and unexpectedly high stability of RNA under certain conditions
[1–3], which has again brought up the topic of RNA in forensic re-
search recently. Applications of RNA quantification in determining
the wound age and postmortem interval as well as identification of
body fluids have been reported [4–6]. For forensic pathologists, the
most attractive area is the diagnosis of the cause of death and eval-
uation of the morphological and functional changes of cells and or-
gans during the process immediately proceeding death. A death
process involving a functional mechanism occurring rapidly can
barely produce morphological changes in visible findings, whereas
the functional status of a cell is displayed in its mRNA pattern.
* Corresponding author. Tel.: +81 6 6645 3767; fax: +81 6 6634 3871.
E-mail address: zhaodong99@hotmail.com (D. Zhao).
down-regulations of gene expression that can occur rapidly within
minutes [7–9]. Therefore, analyzing the mRNA pattern of cells in
postmortem samples is believed to offer insight into pathological
mechanisms leading to death (Fig. 1). With respect to this, this re-
view discusses postmortem mRNA analysis for practical applica-
tions in forensic pathology, by offering an overview of our
previous and ongoing studies.
2. Gene expression of hypoxia-inducible factor 1 and its
downstream factors
In the animal kingdom, a global hypoxia response system med-
iated by the transcriptional activator HIF-1 functions as a key reg-
ulator of O2 homeostasis that facilitates both O2 delivery and
adaptation to O2 deprivation [10,11]. The number of known HIF-
1 target genes continues to increase rapidly, now being more than
40, and the established roles of the encoded proteins provide a
molecular basis for biological phenomena, including erythropoie-
sis, vascularization, and energy metabolism [12–14]. Tissue ische-
mia and systemic hypoxia are the most common pathological
changes involved in the processes of various diseases and deaths,
and, therefore, we chose these hypoxia-responsive factors as pri-
mary candidate targets in our studies.

1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.legalmed.2009.01.066
S44 D. Zhao et al. / Legal Medicine 11 (2009) S43–S45
Physiological status
Insult of fatal trauma or disease
Pathophysiological reactions Alteration of mRNA
pattern in cells
Death Interpretation
Postmortem degradation Postmortem mRNA
analysis
Decomposition
Fig. 1. Postmortem mRNA analysis for diagnosing the cause and process of death.
3. Influence of postmortem changes on mRNA analysis in
autopsy cases
Prior to practical application in autopsy casework, a pilot exper-
imental study was performed to establish quantitative assays of
hypoxia-responsive factors including hypoxia-inducible factor 1
alpha (HIF-1a), vascular endothelial growth factor (VEGF), and
erythropoietin (EPO) mRNAs, and to investigate the postmortem
degradation profiles of these mRNAs in forensic autopsy materials.
Relative quantification of HIF-1a, VEGF, and EPO mRNAs based on
the TaqMan real-time RT-PCR were performed using autopsy tissue
specimens from the kidney after preservation at room temperature
for various storage times. HIF-1a and VEGF mRNAs gradually de-
graded in patterns similar to glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) mRNA used as an endogenous reference.
Accordingly, the relative quantification of HIF-1a/GAPDH and
VEGF/GAPDH changed little up to 48 h postmortem in the kidney.
However, the status was different for EPO mRNA, with high-level
stability regarding postmortem degradation and a marked post-
mortem time-dependent increase in the EPO/GAPDH ratio for au-
topsy tissue samples [15]. Similar postmortem degradation
profiles of these mRNAs were also found in other organs and tissue
samples. This pilot study suggested the potential for applying
quantitative analyses of mRNA transcripts to autopsy materials,
and indicated the significance of investigating degradation profiles
prior to carrying out the relative quantification of target mRNAs in
autopsy materials. Besides the postmortem interval, the age and
gender of subjects usually show an insignificant or mild influence
on the relative quantification of mRNA in adult autopsy cases. In-
fancy cases, although without a detailed investigation based on
large-scale studies, might exhibit pathophysiological particularity
compared with adult cases. Thus the cut-off value of mRNA levels
for diagnosis should be set with this in mind.
4. Applicability of postmortem mRNA analysis for the diagnosis
of the cause and mechanism of death
Following the pilot study concerning the influence of postmor-
tem changes on mRNA quantification, we investigated HIF-1a, EPO,
and VEGF mRNA expressions with regard to the cause of death in
autopsy kidneys. Protocols of mRNA assays mainly referred to
studies previously performed in our institute on the postmortem
molecular biological analysis of pulmonary surfactant-associated
protein (SP-A) [16,17].
Relative quantifications of HIF-1a, EPO, and VEGF mRNAs were
performed using tissue specimens taken at consistent sites of the
renal cortex on both sides. Both HIF-1a and EPO mRNA expression
levels were significantly lower in drowning cases. VEGF mRNA
showed higher expression levels for acute myocardial infarction/
ischemia (AMI), acute blunt/sharp instrument injuries, and aspira-
tion, whereas it was lower for neck compression (strangulation/
hanging), drowning, fire fatalities with a higher blood carboxyhe-
moglobin (COHb) level (>60%), peracute injury death, and gastroin-
testinal hemorrhage. The quantitative assays of renal HIF-1a, EPO,
and VEGF mRNA expressions are potentially helpful to investigate
the pathophysiology of death involving acute circulatory failure
[18].
Further investigation of VEGF together with glucose transporter
1 (GLUT1) regulated by HIF-1 was performed in the lung and skel-
etal muscle as well as in the kidney, to test the hypothesis that
mRNA quantification of these factors in various autopsy tissues
could have diagnostic significance for investigating the pathology
of death, especially after traumatic injury. Characteristic results
were found in blunt injury cases: both GLUT1 and VEGF mRNAs de-
creased in the lung but increased in the skeletal muscle depending
on survival time. In the kidney, subacute deaths showed higher
GLUT1 mRNA levels compared with acute deaths from blunt injury,
but no significant change was found for VEGF mRNA. Immunohis-
tochemistry showed visually predominant GLUT1 immunoreactiv-
ity in the renal cortex for cases with a longer survival time,
coincident with the results at the mRNA level. Tissue-specific dif-
ferences in mRNA quantification of GLUT1 and VEGF shed light
on tissue ischemia/hypoxia and subsequent tissue-dependent
pathophysiological changes leading to death after injury [19].
A comparison between infantile and adult deaths from mechan-
ical asphyxia and pulmonary infection suggested significant differ-
ences: adult cases showed lower VEGF mRNA levels in the lung and
higher GLUT1 mRNA levels in the skeletal muscle for pulmonary
infection than for asphyxiation, while such differences were not
detected in infants. Compared with adults, infantile cases showed
a higher VEGF mRNA level in the lung for pulmonary infection,
and higher GLUT1 and VEGF mRNA levels in the skeletal muscle
for asphyxiation. For pulmonary infection, both mRNA levels in
the skeletal muscle were similar in infantile and adult cases. These
findings indicate a similar death process due to pulmonary infec-
tion and asphyxiation in infantile cases; thus, it might be essen-
tially difficult to distinguish these causes of death. The death
process of pulmonary infection and mechanical asphyxia might
differ between infantile and adult cases.
Another study investigated the immunohistochemical distribu-
tions and mRNA expressions of myocardial HIF-1a, EPO, and VEGF
in cardiac deaths. HIF-1a, EPO and VEGF mRNA levels, as measured
by real-time RT-PCR, which showed localized elevations related to
AMI lesions, are indicative of short and substantial durations of
myocardial ischemia, respectively. Combined analyses may not
only be useful for investigating the site, phase, and severity of
acute myocardial ischemia, but also the severity of chronic ische-
mic stress [20].
5. Experimental studies using a rat model of fixed-volume
hemorrhage
Our previous studies have revealed the diagnostic significance
of mRNA quantification in death investigation of medicolegal au-
topsy, especially for injury cases. A rat (SD) hemorrhage model
was adopted in one of our ongoing studies for a comparison with
the results of human autopsy samples.
The right femoral artery of rats was cannulated with a polyeth-
ylene tube for blood withdrawal under anesthesia. Blood was with-
 D. Zhao et al. / Legal Medicine 11 (2009) S43–S45
drawn into a syringe until the volume reached 24 ml/kg (ca. 30% of
whole blood), and then it was maintained for 60 or 120 min. Con-
trol rats were anesthetized and treated as unchallenged controls.
Following the decapitation of rats, tissue specimens were collected.
The animal study supported the results of autopsy materials,
showing a significant increase of VEGF mRNA levels in skeletal
muscles of the bilateral forelimbs of hemorrhagic rats normalized
against endogenous references of GAPDH and b-actin. Further-
more, postmortem degradation profiles of mRNAs were also as-
sessed in this animal study. Postmortem degradation patterns of
VEGF and GAPDH mRNAs showed tissue-specific differences in
the lung and skeletal muscle. However, in individual samples, dif-
ferent mRNAs showed similar degradation patterns; therefore, the
postmortem changes showed no significant influence on the rela-
tive quantitative analysis of VEGF in the lung and skeletal muscle.
In addition, also using this rat model, our ongoing studies on cell
growth and apoptosis-related factors revealed that the mRNA level
of c-myc showed an increase in the early postmortem period,
which was considered to possibly represent a supervital reaction
of cells in the lung. These results verified a part of previous findings
in autopsy cases and indicate the usefulness of animal studies for
investigating potentially useful mRNA targets for practical applica-
tion in medicolegal autopsy casework.
6. Conclusion
Postmortem degradation patterns of each mRNA are tissue-
dependent; in individual tissue samples, most mRNAs show similar
degradation patterns. Therefore, the relative quantification of
mRNAs may be reliable within 48 h postmortem; however, some
special states possibly representing supervital reactions should
be paid attention to. The postmortem mRNA quantification of hy-
poxia-responsive factors is useful in the diagnosis of the cause
and mechanism of death, to support and reinforce morphological
evidence.
Forensic research in this field is at its very beginning, with its
applicability to human postmortem tissues still to be verified,
but our studies suggest that it holds promise as an interesting area
relevant for both forensic and clinical medicine [21]. Research in
this field will lead to cooperation between medical professionals
and molecular biologists, which is necessary for standard and reli-
able death investigations and mRNA analyses. Animal experiments
will be useful in selecting ideal markers and comparing with au-
topsy studies, although the status may somewhat differ between
human beings and animals. The combination of high-throughput
microarray and real-time PCR techniques will be effective in profil-
ing tissue-specific gene expression and constructing an mRNA
database of diagnostic significance. In theory, quantitative analysis
of mRNA from autopsy samples based on high-throughput assays
could be compared with a database and provide an evidence-based
diagnosis even without specific morphological findings.
Conflict of interest
The authors have no financial conflict of interest.
S45
Acknowledgements
This review was supported in part by Grants-in-Aid for Scien-
tific Research from the Japan Society for the Promotion of Science
and the Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan (Grant Nos. 11670425, 12470109, 08307006,
15390217, and 15590585).
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