2. • mRNA (messenger RNA) and microRNAs (miRNAs) are both
promising tools for body fluid identification and estimating
bloodstain deposition time in forensic science due to their
unique properties and roles in gene expression:
3. • These are considered as confirmatory tests that conclusively identify a
body fluid.
• mRNA profiling is based on the premise that each single tissue type is
comprised of cells that have a unique transcriptome or gene expression
(i.e., mRNA) profile.
• A number of markers have been identified for the forensically most
relevant body fluids, that is, blood, saliva, semen, vaginal secretions,
menstrual blood, and sweat.
• mRNA is notorious for its rapid postmortem and in vitro decay, due to
ubiquitously present RNases.
• This was expected to be especially problematic as biological stains from
casework are often challenged by moisture, UV light, temperature, and
suboptimal environmental pH, thus potentially resulting in mRNA of
insufficient quality for analysis.
• Quite unexpectedly, however, a high stability of mRNA in dried stains, even
from old and compromised samples, has been reported
4. • Recently, there has been an explosion of interest in a class of small
noncoding RNAs, miRNAs, whose regulatory functions in various
developmental and biological processes have been identified.
• Numerous studies have indicated that miRNAs are also expressed in a
tissue-specific manner and therefore may be used to identify body
fluids.
• Due to their small size (18–24 bases in length), miRNAs may be more
stable than mRNAs and therefore more suitable for use with
degraded and environmentally compromised samples frequently
encountered in forensic casework.
5.
6. • Distinguishing Casual Contact from Sexual Assault: In your
example, the suspect claims that the presence of the victim's
DNA in their vehicle is due to casual contact because the victim
had ridden in the car multiple times. However, if forensic
analysis can conclusively identify the source of the DNA as the
victim's vaginal secretions, it becomes much more challenging
for the suspect to attribute the DNA to casual contact. This is
because vaginal secretions are not typically exchanged during
ordinary, non-consensual interactions.
7. • Supporting the Victim's Account: Identifying the DNA source
as vaginal secretions provides additional support for the victim's
account of the sexual assault. It strengthens the victim's
credibility and can help establish a more compelling narrative in
court.
8. mRNA for Body Fluid Identification:
1.Specific Expression Profiles: Different body fluids have
distinct mRNA expression profiles. This means that the mRNA
present in blood differs from that in saliva, semen, vaginal fluid,
or sweat. By analyzing these unique mRNA profiles, forensic
scientists can determine the source of a body fluid found at a
crime scene.
2.High Sensitivity: mRNA analysis can be highly sensitive,
allowing for the detection of trace amounts of mRNA in a stain,
even when the stain is old or degraded. This sensitivity is critical
in forensic investigations where samples may be limited or
degraded over time.
9. Messenger RNA
• mRNA is a single-stranded nucleic acid, consisting of four kinds of
nucleotides: uracil, guanine, adenine, and cytosine. mRNA is
transcribed from a DNA template and carries coding information to
the ribosomes, where it is translated into a protein.
10. • During transcription, RNA polymerase II makes a complementary copy
of a gene from DNA to pre-mRNA.
• Eukaryotic pre-mRNA undergoes extensive processing: 50 cap
addition, splicing, editing, and polyadenylation.
• The 50 cap is critical for recognition by the ribosome and protection
from RNases.
• During splicing, certain stretches of noncoding sequences (introns)
are removed.
• In some instances, an mRNA is edited by changing its nucleotide
composition. The poly(A) tail at the 30 end helps to protect mRNA
from degradation by exonucleases.
• Polyadenylation is also important for transcription termination,
export of the mRNA from the nucleus, and translation.
11. • The mature mRNA is exported from the nucleus to the cytoplasm,
where it is translated into protein by the ribosome.
• During translation, the sequence of nucleotides in the mRNA
molecule is read in sets of three nucleotides (called codons), each of
which codes for a specific amino acid.
• The resulting polypeptide will later fold into an active protein. mRNA
is degraded by several decay pathways. .
12. Detection Technology
• mRNA profiling includes three main steps: RNA extraction, reverse
transcription (RT), and amplification/detection.
• Total RNA and DNA can be coextracted from the same stain, either
manually or with an extraction kit.
• The RNA extract should be cleaned from residual DNA using a DNase
treatment. It is beneficial to quantify the RNA and put a defined
amount into the RT reaction to avoid false positive and negative
signals.
• In the RT reaction, mRNA is reverse-transcribed into complementary
DNA (cDNA) using random primers. This step is essential because
forensic laboratories primarily work with DNA analysis
techniques.
13. • cDNA is then amplified with fluorescence-labeled, tissue-specific
primers (end-point polymerase chain reaction – PCR) and amplicons
are separated and detected with capillary electrophoresis.
• Alternatively, cDNA may be analyzed using real-time quantitative PCR
(qPCR).
• mRNA markers can be combined in multiplexes, for end-point PCR as
well as qPCR, which amplify several markers for one or several body
fluids in a single PCR. Coextracted DNA can be analyzed according to
standard Short tandem repeat (STR) protocols
14. Tissue-Specific mRNA Markers
• A number of mRNA markers have been identified for the forensically
most relevant body fluids, that is, blood, saliva, semen, vaginal
secretions, menstrual blood, and sweat. Table 2 lists a selection of
commonly used markers that have been reported as fluid-specific
15.
16.
17. • The evaluation of mRNA markers for forensic use includes testing their
sensitivity and specificity, as well as performance with casework samples.
• The above-mentioned blood, saliva, and semen markers show a high
degree of specificity and no relevant cross-reaction with other human
tissues/body fluids or with other species.
• It has been demonstrated that the corresponding mRNA markers can be
detected in samples as small as 0.001 ml blood, 0.05 ml saliva, and 0.01 ml
semen.
• The analysis of sperm and seminal plasma markers allows for the
differentiation between samples from aspermic (absence of sperm in
semen) men and normospermic men (with normal sperm production).
• The vaginal markers mucin 4 (MUC4) and human beta-defensin 1 (HBD1)
cross-react with buccal cells (i.e., saliva) and are therefore only of limited
use in forensic casework.
18. • . The matrix metalloproteinases (MMP) markers confirm the presence of
menstrual blood, but a minor expression is also found in vaginal cells.
• This complicates the interpretation of a positive MMP result, but could be
overcome by comparing the result to a housekeeping gene and/or the
vaginal secretion markers HBD1 and MUC4, which are expressed quite
constantly and strongly during the whole menstrual cycle.
• The blood markers are rather weak in menstrual blood samples, because
whole blood accounts for only 30–50% of the total flow in most women.
Additional mRNA markers have been suggested for the identification of
vaginal secretion, menstrual blood, and sweat, but have not yet been
extensively tested for forensic use.
• To date, no mRNA markers for the identification of urine are available,
probably due to the limited number of cells and/or low mRNA expression
in dried urine stains.
19. • Since casework material is often limited, an important advantage of
body fluid identification by mRNA profiling is the possibility of
simultaneously isolating RNA and DNA from the same stain
• The mRNA/DNA coextraction strategy is a reliable, confirmatory, and
time-saving option for the identification of body fluids in forensic
casework that requires less sample consumption and is also
compatible with the current DNA analysis methodology.
20. MicroRNA
• miRNAs are a class of small noncoding RNAs, 18–24 nucleotides in
length, which regulate many cellular processes at the
posttranscriptional level.
• The human genome encodes over 1000 miRNAs
21. miRNAs for Body Fluid Identification:
1.Stability and Persistence: miRNAs are small non-coding RNA
molecules that are remarkably stable in various environmental
conditions. They can remain intact even in degraded samples.
This stability makes miRNAs suitable for identifying body fluids
in challenging forensic samples.
2.Tissue-Specific miRNAs: Just like mRNA, different body fluids
contain unique miRNA profiles. Certain miRNAs are specific to
particular tissues or body fluids, making them valuable markers
for identification. For example, specific miRNAs might be found
in blood but not in saliva, or vice versa.
22. Tissue-Specific miRNA Markers
• Over 16 000 miRNAs, across 153 species, have been entered into the
miRBase,
• Numerous miRNAs have been reported to be tissue-specific and could
be used to identify the body fluid origin of forensic biological stains.
• Human miRNA markers were screened in forensically relevant body
fluids (blood, semen, saliva, vaginal secretion, menstrual blood) by
two different research groups using different approaches, resulting in
differing body fluid-specific miRNA markers (Table 3).
25. • The first study on miRNAs and forensic BFID was conducted in 2009
by Hanson et al. The authors examined 452 miRNAs in five forensic
relevant body fluids (semen, venous blood, saliva, menstrual blood,
and vaginal secretion) with qRT-PCR. They identified a panel of nine
differentially expressed miRNAs with a high degree of specificity in
each body fluids. In particular, miR-451a and miR-16 were higher in
venous blood; miR-412 and miR-451a were identified in menstrual
blood, miR-135b and miR-10b in semen; miR-205 and miR-658 in
saliva while miR-124a and miR-372 were identified in vaginal
secretions. Furthermore, the small quantity of total RNA (50
picograms) needed for miRNA analysis underlined the usefulness of
these markers in case of a low amount of forensic samples
26. • One year later, Zubakov et al. investigated a set of 718 miRNAs in five
body fluids with microarray. Two miRNAs for venous blood (miR-144
and miR-185) and two for semen (miR-135a and miR-891a) were
confirmed as differentially expressed with qRT-PCR, suggestive to be
useful for venous blood and semen identification.
27. • Courts and Madea performed an array that examined 800 miRNAs in
venous blood and saliva and validated six candidate miRNAs. They
proposed miR-126, miR-150, miR451a for the identification of venous
blood and miR-200c, miR-203, miR-205 for saliva
28. • Screening a total of 754 miRNAs, Wang et al. proposed five body
fluid-specific miRNAs, whose detection were highly sensitive: miR-16
and miR-486 for venous blood, miR-214 for menstrual blood, and
miR-888 and miR-891a for semen. They highlighted three new miRNA
markers (miR-486, miR-888, and miR-214) but did not identify specific
miRNAs for saliva and vaginal secretions
29. • Two years later, the same authors aiming to find a strategy for saliva
identification, analyzed eight potential saliva miRNAs selected. Only
three miRNAs (miR-200c-3p, miR-203a, miR-205-5p) showed different
expression patterns among these body fluids.
• In particular, miR-200c-3p was higher in vaginal secretions compared
to other body fluids; miR-203a was higher in menstrual blood, vaginal
secretions, and saliva and not present in venous blood; miR-205-5p
was higher in menstrual blood and vaginal secretions compared with
saliva and semen, but not detected in venous blood.
30. • A year later Sauer et al. published a comprehensive study based on a
thoroughly validated qRT-PCR procedure. Four miRNAs (miR-144-3p,
miR-891a-5p, miR-203a-3p and miR-891a-5p) were reported useful
for BFID but only miR-891a-5p was confirmed as truly semen specific.
• The authors proposed a decision algorithm to detect each body fluids
employing few markers to simplify the analysis: miR-891a-5p for
semen identification, miR-144-3p to discriminate blood from non-
blood samples, miR-144-3p and miR-203a-3p to distinguish between
venous and menstrual blood, miR-203a-3p and miR-124-3p to
differentiate between saliva and vaginal secretions
31. • In a study conducted on 15 miRNAs (selected from the literature) in
six body fluid samples (including skin), Sirker et al. validated four
candidate internal controls (miR26b, miR92, miR144, and miR484) as
the most stably expressed across the samples analyzed [42].
• miR-451a was a strong biomarker for all blood types identification.
• The combination of miR-451a and miR-943 could distinguish blood
from other body fluids while miR-943 was useful to separate
menstrual blood from skin.
• miR-374 alone could discriminate semen from blood samples.
• miR-203 could separate vaginal secretion from saliva, semen, and
blood
32.
33. mRNA for Bloodstain Deposition Time:
• Degradation Over Time: mRNA degrades predictably over
time, following a known degradation pattern. This degradation
can be used to estimate the age of a bloodstain. As mRNA
molecules break down, the analysis of their degradation pattern
can provide insights into when the stain was deposited.
34. miRNAs for Bloodstain Deposition
Time:
• Stability in Degraded Samples: miRNAs are more stable than
mRNA and can still be detected in older or degraded
bloodstains. This makes them useful for estimating the
deposition time of bloodstains, even when traditional methods
might not work due to degradation.
35. • They bind to complementary sequences on target mRNA transcripts, usually
resulting in translational repression and gene silencing.
• Most miRNA genes are found in intergenic regions or introns and are transcribed
by RNA polymerases to primary miRNA (pri-miRNA).
• Pri-miRNAs are processed by the enzyme ‘Drosha,’ resulting in long hairpin-
containing precursor miRNA (pre-miRNA).
• ‘Exportin-5’ transports pre-miRNA from the nucleus to the cytoplasm.
• An RNase called ‘Dicer’ cleaves the pre-miRNA and produces double-stranded
mature miRNA.
• Argonaute protein (Ago2) and Dicer together form the ribonucleoprotein RNA-
induced silencing complex (RISC).
• One strand of the mature miRNA is incorporated in the RISC and guides the
complex to its RNA target by interacting with the three prime untranslated
regions (30 UTR) of the mRNA.
• miRNA-mediated gene regulation depends on the complementarity between
miRNA and target mRNA:
36. • If there is perfect complementarity Ago2 can cleave the mRNA and
lead to direct mRNA degradation; if there is only partial
complementarity, the silencing is achieved by preventing translation.
• This mechanism implies that any given miRNA can bind to a broad
spectrum of different mRNAs and any given mRNA can be bound by
several miRNAs, thereby expanding miRNA regulatory potential.
• In addition, miRNA regulation can fine-tune and optimize protein
levels using a ‘rheostat’-like mechanism.
