Legal Medicine 47 (2020) 101727

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

Simultaneous DNA and RNA profiling in a case of sexual assault in a 3-year- T

old child: Forensic genetics solves the crime
⁎
V. Cortellini , G. Brescia, N. Cerri, A. Verzeletti
University of Brescia – Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, Forensic Medicine Unit, Brescia, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: DNA profiling can identify an individual from a sample of biological material but it does not reveal what body
DNA/RNA profiling fluid or tissue source the DNA profile originated from. In many cases it is important to know from what body
Body fluid identification fluid or tissue the DNA profile originated in order to provide crucial information necessary to the investigation,
Child sexual assault especially in cases where the victims are not able to give information about the dynamics of the event. For this
purpose messenger RNA (mRNA) analysis has been shown to be a suitable method for the identification of body
fluids, resulting in a trend to overcome the conventional approaches. Here we present the first report about case
regarding a three-year-old child supposedly victim of a sexual assault with digital penetration. Thanks to the use
of the combined DNA profiling and RNA analysis it was possible to demonstrate the sexual assault suffered by the
victim.

1. Introduction
Biological stains are commonly encountered in forensic casework;
DNA profiling to identify the subject to which a biological trace belongs
is a routine practice worldwide, and this represents potentially crucial
‘source level’ information for investigations [1,2]. However there are
many cases where it is important to know not only who is the source of
the sample, but also from what body fluid or tissue the DNA profile
originated. In fact the context and relevance of the DNA profiled in the
case may depend on the identification of the body fluid from which it
has originated [3]. Particularly, in the hypothesis of a sexual assault,
body fluid stains recovered on the victim/suspect’s body are important
evidence for forensic investigation: the ability to determine the tissue
source of forensically relevant biological fluids could provide crucial
information necessary to the investigation, especially in cases where the
victims are not able to give information about the dynamics of the event
[4,5]. Moreover, next to DNA typing results, knowledge about the
cellular origin of crime-related biological stains can be of great im-
portance for the reconstruction of the events at a crime scene [6]. In
instances of sexual assault with a foreign object or digital penetration,
the identification of vaginal secretions (VS) transferred to such objects
or the perpetrator might be critical in establishing the circumstances of
the assault. The ability to definitively identify vaginal epithelia or their
secretions (both referred to hereafter as VS) as the source of a DNA
profile would significantly aid the investigation of sexual assaults, since
many of these cases result in the transfer of VS from the victim to a
person or object.1
Conventional methods of body fluid identification, protein or en-
zyme based, are presumptive in nature and not always human specific;
furthermore, they are not always applicable if the stain is very small. In
recent years, messenger RNA (mRNA) analysis has been shown to be a
suitable method for the identification of body fluids, resulting in a trend
to overcome the conventional approaches. Expression of specific mRNA
varies among cell types, therefore analysis of these makers can be used
to determine the presence of specific biological fluid within a sample
[7]. Tissue-specific mRNA detection offers crucial advantages in for-
ensic casework: high sensitivity due to the possibility of PCR amplifi-
cation, high specificity due to the pattern of gene expression, unique for
the functional status of cells and organs, simultaneous DNA isolation
without material loss, using extraction methods that concurrently iso-
late RNA and DNA from the same stain extract, and, last but not least,
the mRNA stability in forensic stains [8]. mRNA profiling has now
evolved to a multiplex PCR platform, thus providing information about
multiple gene expression and could easily be combined with DNA
profiling from the same exact sample, using a co-extraction method
[9,10]. In this way, analysis on the mRNA extracts will yield informa-
tion regarding the origin of the stain, and the DNA analysis will reveal
the donor’s identity.

⁎
Corresponding author at: Piazzale Spedali Civili 1, 25123 Brescia, Italy.
E-mail address: venusia.cortellini@unibs.it (V. Cortellini).

https://doi.org/10.1016/j.legalmed.2020.101727
Received 8 July 2019; Received in revised form 2 October 2019; Accepted 1 June 2020
Available online 06 June 2020
1344-6223/ © 2020 Elsevier B.V. All rights reserved.
V. Cortellini, et al.
Fig. 1. Little lesion in correspondence of the right labia minora.
2. Case report
A little girl of about three years old was taken to the emergency
room by her parents because she complained of vulvar burning and
blood loss during urination.
The parents told the doctors that in that same afternoon the child
had been under the sole care of a family friend.
On physical examination the little girl appeared in good general
condition, with no signs of recent trauma on the skin; she didn’t answer
any questions from the doctors.
At the gynecological examination, the inspection of the external
genitalia showed regular vulva with intact skin except for two reddish
excoriations, one of about 2–3 mm in correspondence of the right la-
bium minus and one of about 5 mm in correspondence of the left labium
minus (Fig. 1). A small reddish point spot lesion was also observed on
the inner face of the right labium minus. Nothing was reported in the
anal region. A vulvar swab was made for subsequent laboratory in-
vestigations. A buccal swab was collected in order to extrapolate the
child’s genetic profile.
Meanwhile, the police arrested the man who had spent time with
the little girl, therefore suspected of having abused her.
Ten fingernail swabs were made, one for each suspect’s finger,
numbered sequentially from one to five (thumb to little finger) for both
right (R) and left (L) hand. Moreover, two absorbent paper sheets were
used to rub the suspect’s dorsal surface of both hands, namely Right
Hand (RH) and Left Hand (LH).
A buccal swab was also collected in order to extrapolate the man’s
genetic profile.
3. Materials and methods
3.1. Sample collection
The vulvar swab and the child’s buccal swab, as well as all the
samples taken from the suspect (collected by scientific police and for-
ensic pathologist) were delivered to the Forensic Genetics Laboratory
for analysis.
Macroscopic observation of the vulvar swab showed a point blood
spot, whose nature was confirmed through the Combur Test.
3.2. DNA extraction and RNA/DNA co-isolation
Suspect and child’s buccal swabs DNA extraction was made using
the classical method of Chelex® 100 (Biorad, Richmond, CA, USA) [11],
while genetic analyses on the vulvar swab, the fingernail swabs and the
blotting papers were performed using the AllPrep™ DNA/RNA Mini Kit
(Qiagen, Hilden, Germany), according to the manufacturer’s
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Legal Medicine 47 (2020) 101727
instructions. Samples were denatured using 350 μl of RLT Plus Buffer
and 6.9 μl of DTT (Dithiothreitol) 2 M and incubated for 3 h at 56 °C to
improve the extraction.
The lysates were transferred to an AllPrep DNA Spin Column to
separate DNA from RNA. The RNA containing flow-through was pro-
cessed first because of the fragile nature of RNA, while the DNA col-
umns were stored at 4 °C until further processing.
3.2.1. DNA
3.2.1.1. DNA purification. The DNA bound to the AllPrep DNA Spin
Column membrane was eluted with Elution Buffer (EB), according to
the manufacturer’s instructions.
3.2.1.2. STR profiling. DNA profiling was performed using the
commercial AmpFℓSTR™ Identifiler™ Plus kit [12] (Applied
Biosystems, Foster City, CA, USA); PCR reactions took place in a
GeneAmp® 9700 Gold Plate (Applied Biosystem, Foster City, CA,
USA), following the manufacturer’s instructions.
All the samples were also amplified using the YFiler™ Plus
Amplification Kit [13] (Applied Biosystems, Foster City, CA, USA),
which allows to amplify loci present on the Y chromosome, present only
in male subjects.
3.2.2. RNA
3.2.2.1. DNAse treatment. The removal of DNA contaminant was
performed with the Turbo DNA-Free (Invitrogen). Purified RNA was
heated with 0.1 vol of DNase Turbo Buffer 10x and 1 μl of Turbo DNase
at 37 °C for 30 min. The DNase inactivation was performed adding
0.1 vol of DNase Inactivation Reagent for 5 min at room temperature.
3.2.2.2. RNA reverse transcription. RNA samples were submitted to
Reverse Transcription (RT) using SuperScript™ IV First-Strand
Synthesis System (Invitrogen), according to the manufacturer’s
instruction.
3.2.2.3. cDNA multiplex polymerase chain reaction. A 19-Plex PCR
containing primers for three blood markers (HBB, CD93, ALAS2),
three saliva markers (HTN3, STATH, BPIFA1), three sperm markers
(KLK3, SEMG1, PRM1), three vaginal mucosa markers (CYP2B7P1,
MUC4, MYOZ1), three menstrual secretion markers (MMP7, MMP10,
MMP11), two skin markers (CDSN, LCE1C), as well as for two
housekeeping genes ACTB and 18S-rRNA, was performed [6,14].
In the multiplex PCR, 3.75 μl cDNA was amplified in the presence of
6.25 μl Master Mix 2x (Qiagen, Hilden, Germany) and 2.5 μl Primer Mix
5x, resulting in total volume of 12.5 μl. The PCR was performed in a
GeneAmp® 9700 Gold Plate (Applied Biosystem, Foster City, CA, USA)
with the following cycling conditions: 95 °C for 15 min, 33 cycles of
94 °C for 20 s, 60 °C for 30 s, 72 °C for 40 s, and a final soak at 60 °C for
45 min.
3.2.2.4. PCR purification. The MinElute™ PCR Purification Kit (Qiagen,
Hilden, Germany) was used to purify the cDNA from primers,
nucleotides, enzymes, mineral oil, salts, and other impurities.
3.3. Capillary electrophoresis
The amplified cDNA and DNA fragments were detected with a 3500
Genetic Analyzer instrument (Applied Biosystems, Foster City, CA,
USA) and the electropherograms were analyzed using the dedicated
software GeneMapper ID-X v1.4 (Life Technologies, Carlsbad, CA,
USA).
3.4. Statistical analysis
DNA mixtures statistical analysis was performed with LRmix Studio
Software [15], an expert system dedicated to the interpretation of
V. Cortellini, et al.
forensic DNA profiles, with a particular focus on complex DNA mix-
tures. LRmix Studio enables measuring the probative value of (auto-
somal STR-based) forensic DNA profile.
4. Results
4.1. DNA profiling
The victim and the suspect’s genetic profiles were obtained from the
analysis of their buccal swabs. In the DNA extracted from the vulvar
swab no mixed profiles were found: the electropherogram corresponded
perfectly with the victim’s profile. Furthermore the Y chromosome
analysis didn’t give any result.
The right thumb fingernail swab (IR) showed a genetic profile
corresponding to the suspect; the fingernail swabs namely IIR, IIIR and
IVL, as well as the blotting papers (LH and RH), revealed a balanced
mixture between the suspect and the victim, in which both the con-
tributors were equally present. It was possible to discriminate allelic
peaks from stuttering peaks thanks to the position of the latter with
respect to the former, because the stutter peaks was immediately before
a real peak. Secondly, we compared the profile of the mixture with the
suspect and victim’s profiles (Fig. 2). The IL and IIL fingernail swabs
showed an incomplete mixture profile in which the victim represented
the major contributor and the suspect the minor contributor (Fig. 3). In
four fingernail swabs (IIIL, VL, IVR and VR) no profile was detected.
4.2. RNA profiling
The mRNA analysis was performed in order to reveal the nature of
the traces on the fingernail swabs and the pads of adsorbent paper used
to blot the suspect’s hands.
The vulvar swab was not further investigated since male biological
material was not identified (no mixed STR profile detected nor Y profile
found), as well as five fingernail swabs since STR analysis didn’t give
any results or showed only the suspect’s genetic profile.
The right thumb fingernail swab (IR) mRNA analysis showed only
skin markers (CDSN and LCE1C); vaginal mucosa (MUC4 and
CYP2B7P1), and skin (CDSN and LCE1C) markers were observed in IVL,
IIR and IIIR fingernail swabs; IL and IIL fingernail swabs showed va-
ginal mucosa (MUC4 and CYP2B7P1) and saliva (STATH) markers.
The mRNA examination of the blotting paper relative to the dorsal
Fig. 2. Electropherogram of DNA profiling of blotting paper relative to the dorsal surface of the suspect’s left hand (LH).
3
Legal Medicine 47 (2020) 101727
surface of the suspect’s left hand (LH) showed vaginal mucosa (MUC4
and CYP2B7P1), saliva (STATH), blood (HBB) and skin (CDSN) markers
(Fig. 4). Vaginal mucosa (MUC4 and CYP2B7P1) and blood (HBB)
markers were observed in the sample obtained from the suspect’s right
hand blotting (RH). Both housekeeping markers (ACTB and 18S-rRNA)
were present in all samples, thus demonstrating the good efficiency of
the analysis.
The results of DNA and RNA profiling for each specimen are re-
ported in Table 1.
4.3. Statistical analysis
It was calculated, for the seven mixtures found, the probability that
they resulted from the victim and the suspect’s profiles. The Likelihood
Ratio (LR) obtained ranged from 4.3135E108 and 4.3317E1018, such
high values that strongly support the hypothesis that these mixtures
derive from the contribution of the two subjects involved.
5. Discussion
The case reported regards a three-year-old child was taken to the
emergency room by her parents because she was supposedly the victim
of a sexual assault with digital penetration. Given her young age, she
was not able to provide information concerning the dynamics of the
event during which she suffered vaginal lesions.
The identification of specific body fluids was shown to be useful in
reconstructing criminal events, especially sexual assaults, where the
victims cannot provide useful information for the investigation, such as
babies, unconscious victims or persons with mental retardation.
For this purpose, in addition to DNA profiling, we have analyzed the
available traces with mRNA multiplex, in order to identify the tissue of
origin of the biological material.
Given that circumstantial data indicated that a family friend had
spent time alone with the little child, the purpose was to discriminate
between the possibility that the assault had left vaginal secretions on
the suspect’s hands under the prosecutor’s hypothesis, or that the pre-
sence of the victim’s genetic profile on his hands was due to a simple
normal skin contact, thus not related to a sexual assault under the de-
fense hypothesis.
Thanks to the use of the combined DNA profiling and RNA analysis
it was possible to confirm the presence of the victim’s genetic profile on
V. Cortellini, et al. Legal Medicine 47 (2020) 101727

Fig. 3. Electropherogram of DNA incomplete mixture profile of fingernail swabs relative to suspect’s left hand second finger (IIL).

Fig. 4. Electropherogram of the mRNA analysis of blotting paper relative to the dorsal surface of the suspect’s left hand (LH). From the left of the first row the RNA
markers are HBB (blood), STATH (saliva), ALAS 2 (blood), SEMG1 (seminal fluid) and MUC4 (vaginal mucosa). From the left of the second row the RNA markers are
CDSN (skin), MYOZ1 (vaginal mucosa), MMP10 (menstrual blood), MMP7 (menstrual blood), HTN3 (saliva) and CYP2B7P1 (vaginal mucosa). From the left of the
third row the RNA markers are MMP11 (menstrual blood), PRM1 (sperm cell), LCE1C (skin) and CD93 (blood). From the left of the last row the RNA markers are
KLK3 (seminal fluid), ACTB (housekeeping), 18S-rRNA (housekeeping) and BPIFA1 (nasal mucosa).

the suspect’s hand and to identify this biological material as vaginal
secretion, confirming that the man had touched/penetrated the little
child genital area. Besides, the identification of blood marker on the
samples obtained from suspect’s hands blotting was compatible with
the presence of the lesions found on the little child’s labia minora
during the gynecological examination, probably due to digital pene-
tration.
Regarding the MYOZ1 marker, the reason which it has never been
detected can be attributed to the fact that this marker has a lower
sensitivity with respect to the other markers. Therefore, it is more dif-
ficult to detect this marker in challenging samples.
In the LH specimen, only CDSN marker was detected, unlike in the
IR sample where both skin markers (CDSN and LCE1C) were detected.
This result may depend on the type of substrate from which the RNA
was extracted: in fact, IR was a fingernail swab, while LH was a blotting
paper.
In conclusion, the great improvement given by RNA technology to
the forensic genetic laboratory demonstrated that, next to DNA-typing
results, knowledge about the cellular origin of crime-related biological
stains can be of significant importance. This type of molecular in-
vestigation is particularly useful for reconstructing criminal events
where the victim cannot provide information about the dynamics of the
facts. Furthermore, as seen in this case, the possibility of DNA and RNA
co-extraction from the same sample and the ability to multiplex mRNA
markers for the identification of one or more body fluids may overcome
conventional identification methods, thus avoiding sample loss.
The coherence between the DNA and RNA profiling results for an
evidentiary trace can therefore translated to a court report to be used by

4
V. Cortellini, et al. Legal Medicine 47 (2020) 101727

Table 1 English revision.

Results of DNA and RNA profiling.
Specimen STR results RNA markers References

Vulvar swab Victim only NTb [1] E.K. Hanson, J. Ballantyne, Highly specific mRNA biomarkers for the identification
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IIL suspect > victim vagina, saliva ciding which level to address in casework, Sci. Justice 38 (1998) 231–239.
IIIL NDa NTb [3] R.I. Fleming, S. Harbison, The development of a mRNA multiplex RT-PCR assay for
IVL suspect = victim vagina, skin the definitive identification of body fluids, Forensic Sci. Int. Genet. 4 (2010)
VL NDa NTb 244–256.
IR suspect only NTb [4] P. Tozzo, P. Nespeca, G. Spiagarolo, L. Caenazzo, The importance of distinguishing
menstrual and peripheral blood in forensic casework, Am. J. Forensic Med. Pathol.
IIR suspect = victim vagina, skin
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Acknowledgment

The authors thank Dr. Heitor Simoes Dutra Correa for his help in