This document discusses the use of RNA profiling technology to identify the tissue source of trace DNA evidence in sexual assault cases. It notes that current STR DNA profiling cannot identify tissue source, which is important for reconstructing events. RNA profiling using mRNA and miRNA markers has emerged as a promising approach, as different tissues express unique profiles of genes and miRNAs. Several studies demonstrate the ability of multiplex RNA profiling to reliably identify blood, saliva, semen, menstrual blood, skin and other tissues from low copy number DNA samples. The stability of miRNAs makes them particularly suitable for analyzing old, degraded forensic samples. The document concludes that RNA profiling, using techniques like RT-PCR and qRT-PCR, shows potential for resolving many forensic cases by

1. TITLE: Use of RNA profiling technology in era of DNA transfer and persis-
tence in sexual offence cases.
Introduction:
Sexual assault mostly leads to evidence of blood, semen, menstrual blood, saliva, skin
microbes and vaginal secretion in single or mixed profile that can be useful indicators of
sequence of events leading to deposition (Taylor et al., 2014). Some of this evidence
becomes trace DNA/profile as in in child sex assault, victims often hide stained items for
weeks or months before washing or seizures for investigation and this lead low DNA profile
recovery or evidence becomes trace low copy number DNA (Brayley-Morris, Sorrell et al.,
2015). Forensic investigation into such profile leads to many questions like: “individual
source, how is deposited and from which tissue source?”(Raymond et al., 2009, Taroni et al.,
2013). Trace or low copy number DNA is defined as limited profile, invisible or profile below
recommended thresholds with unidentified tissue source in routine analysis through
presumptive test or STR DNA profiling (Romsos and Vallone, 2015). Low copy number (LCN)
DNA (trace DNA) profile could also be obtained from finger nails, finger-marks, skins, wash
clothing or derived semen, diluted blood, dried blood stains, touch marks on surfaces etc.
(van Oorschot et al., 2010).
DNA Transfer and persistence:
Trace DNA is deposited on surfaces either by directly from body tissues, finger-nails, finger-
marks, skin surface contacts (Zoppis et al., 2014) or indirectly through intermediate or
activity such as speaking, coughing and sneezing within the vicinity of an item resulting
transfer of DNA from an individual without contact. DNA is believe to be transferrable and
persisted in crime scenes without the source individual causing such transfers as reveals in
(Brayley-Morris et al., 2015, Helmus et al., 2015) as significant transfer of donor DNA from
skin and epithelial cells successfully were obtained through secondary transfer level. Many
research including Helmus et al., reported that probability of transfer and persistence of
DNA from primary or secondarily sources depends on the nature of the donor, activity, time,
material and surface of the item (Helmus et al., 2015). As (Zoppis et al., 2014, van den Berge
et al., 2016) confirmed that sebum promotes DNA transfer in actual touching, there is
expectation of defence that the DNA profile in sexual case may be due to touching of skin or
secondary transfer and not commission of sexual crime. However, as mentioned in (Goray et
al,. 2010) secondary transfer of low copy number DNA of different biological DNA depends
on the activity, nature of the substrate, moisture content, and the nature of contact between
surfaces, therefore tissue specific analysis is crucial in evaluating the probability of transfer
or event reconstruction.
Current STR profiling
In molecular forensic, linking people, evidence, and crime scenes are often established
through short tandem repeats (STR DNA) profiling which is generally accepted to be unique
for every individual (Goray et al., 2010, Brayley-Morris et al., 2015). STR profiling is still the
gold standard as individual allele has unique differential repeats patterns at specific loci and
single multiplex PCR is essential and advantageous method of amplification (Romsos and

2. Vallone, 2015), however as for analysing trace DNA evidence, this method is not tissue
source identifiable and therefore requires improvements. Although mention in (Zoppis et al.,
2014) that trace DNA or low copy number DNA are fully profile from primary and secondary
transfers, Marshall et al., reveals that short tandem repeat (STR) typing of low copy number
DNA present exaggerated stochastic effects during PCR resulting in heterozygote peak height
imbalance, allele drop out, and increased stutter (Marshall et al., 2015). This means STR
profiling has disadvantages in analysing LCN profile for biomarkers to attribute the source to
a specific tissue which is most challenging issue in sexual assault cases where trace and
mixed evidence are presented.
RNA Profiling:
It is established that each biological tissues have specific genetic expression patterns which
could not be analysed in STR-DNA profiling. Recently, improvement on methods and protein
biomarker expression on tissue specific for LCN DNA are robust (van den Berge et al., 2016)
and Marshal et al., acknowledge that betaine treatment at concentration of 1.25 mol/L
improve the robustness of the amplification and yield of LCN PCR product (Marshall et al.,
2015). Discovery of RNA (mRNA/miRNA) biomarkers that are differentially expressed in body
fluids commonly found at crime scenes (e.g. venous blood, menstrual blood, saliva, and
semen) or skin has contributed to the prospect of forensic investigations (An, Shin et al.,
2012, Wang et al., 2016). RNA-profiling represent an alternative strategy as in Richard et al a
single multiplex mRNA using DNA/RNA co-isolation protocol simultaneously amplify 19
mRNA markers (Richard, Harper et al., 2012). Recent studies (Park et al., 2013) reveal using
whole genome gene expression, mRNA instability was not seen as co-extracted with DNA
achieved significant values for tissue specific proteins, however requirement of larger
sample volume if not co-extracted with DNA makes mRNA not favoured method. The
advantage mentioned by (Lindenbergh et al., 2013, Marshall et al., 2015) that multiplex
analyses of blood, saliva, semen, menstrual blood, vaginal mucosa, general mucosa and skin
with miRNA markers resulted high sensitivity, specificity as full RNA and DNA profiles were
obtained with specific markers of body fluid and skin tissue types (Balding, 2013).
As in Lindenbergh et al., RNA profiling is useful in sexual cases for menstrual secretion,
peripheral blood, vaginal mucosa, semen, touch marks and skin cells as they have analysed
15 different sets with results of specific tissue source profiling (Lindenbergh et al., 2012,
Lindenbergh et al., 2013). Further, same article reveals that using multiplex assay; blood,
saliva, semen, menstrual secretion, vaginal mucosa, general mucosa and skin markers shows
high sensitivity and specificity profiles. This means tissue identification of semen, skin,
bloods and vaginal secretions is achievable in sexual assault (Richard, Harper et al. 2012),
although mucous and vaginal secretions could be difficult due to co-localization and the
cyclic changes. Though RNA profiling is useful, mRNA proves disadvantageous with stability
under unfavourable environmental conditions due to low yield (Richard et al., 2012).
Also due to mixed profile, LCN and trace DNA, there is complexity to the interpretative
process as profile may be of two or more contributors at trace levels (Petricevic et al., 2010).
Furthermore, DNA derived from a single source could be treated as a mixture due to high
stutter peaks and wrongly interpreted as from multiple individuals (Buckleton et al., 2014).
Given the high probability of drop-in, drop-out and increased stutter, estimating the number

3. of contributors can be problematic at any given locus. Amplification bias also cause minor
contributor's alleles to drop out entirely at some loci or cause over-amplification of some
alleles, creating the appearance of a separate contributor. In particular, the increased stutter
seen with trace DNA amplification creates formidable problems for interpretation. Although
there are locus-specific stutter percentage guidelines for standard template amounts and
none exist for trace DNA amounts, there is evidence that forward and backward stutter
increases with increasing allele length within a locus. It is with this recommendation that
mixture interpretation is based on Likelihood Ratio approach for low template mixtures and
the incorporation of an assessment of the probability of allele drop-in and drop-out when
considering a mixture (Biedermann et al., 2014)
Discussion:
In sexual crimes, reliable identification of source and tissue specific profiling is significant to
event reconstruction and contribute toward solving investigation. Recently, investigation into
trace DNA has emphasis on improving methods and biomarkers in order to acquire highly
significant genetic profiles from tiny amounts of low copy number DNA to help identify the
source and tissue type (van den Berge et al., 2016). Information on tissue origin of traces LCN
DNA is mostly determined by RNA profiling on mRNA using DNA/RNA co-isolation protocol as
analysed and simultaneously amplified in (Richard et al., 2012a). Although RNA is an
alternative, mRNA is not as stable as DNA and this necessitates the use of miRNA which has
definitive small structures proprieties and less prone to degradation under unfavourable
environmental (Donfack and Wiley 2015).
The new robust methods of RNA profiling potential using tissue-specific protein expression
markers of miRNA are forensically relevant. The most important advantage of miRNA
profiling is its ability to analyse low copy number, old evidence be analysed in different body
fluids in multiplex reaction providing expression of multiple genes from different tissue
types. This is achieved through reverse transcription endpoint polymerase chain reaction
(RT-PCR) and more importantly real-time quantitative reverse transcriptase-PCR (qRT-PCR)
for body fluid-specific markers as analysed from 200pg-12ng using histatin 3 (HTN3) and
statherin (STATH) for saliva, mucin 4 (MUC4) for vaginal secretions, matrix metalloproteinase
7 (MMP7) for menstrual blood, delta-aminolevulinate synthase 2 (ALAS2) for peripheral
blood, and protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen (Richard et al.,
2012c). A complete profiles and substantial increases in peak heights were reported when
incorporation of a small number of locked nucleic acid (LNA) bases into the mini-STR primers
show an increase amplification success of trace DNA (Williams et al., 2014). Therefore more
stable miRNA markers with none coding region (18-22) nucleotides in length with regulated
gene expression pattern at post-transcriptional level are now robust method as they can
show tissue specific gene expression (Machado et al., 2015). Evaluation and validation of
microRNA (miRNA) biomarkers are potentially better alternative approach in tissue
identification (Richard et al., 2012c) as intrinsically smaller size of miRNA benefits none
degradation under long term environmental conditions compared to mRNA. As reveal by
Donfack and Wiley that miRNA could be extracted from saliva, blood, vaginal fluids, and
menstrual blood using qRT-PCR to the advantage of miRNA expression pattern from low
copy number DNA, trace DNA and old evidence (Donfack and Wiley, 2015). The evaluation
and validation of numerous body fluid specific miRNA biomarkers are potentially increasing
in forensic as 24 participating laboratories in European DNA Profiling Group (EDNAP) using

4. RNA extraction or RNA/DNA co-extraction methods has reliable RNA profiling for body fluid
identification with combined with STR typing technology (Haas et al., 2014). Also to solve
interfering issues, post-PCR purification protocol based on AMPure XP beads that have size-
selective properties were developed (Westen et al., 2013). The increased discussion on
appropriate interpretation for low copy DNA profiles has helped to move the forensic field
forward as more rigorous strategy for interpreting the evidence and presenting appropriate
statistical measures to the courts (Benschop et al., 2015).
CONCLUDING:
STRs DNA profiling individualised source identity successfully without tissue source identity;
therefore progression in technology using robust methods, interpretation, data analysis and
additional molecular markers in order to add value information to the evidence tissue source
is crucial in trace DNA analysis. This means various specialised protein like mRNA, miRNA
with improved techniques like RT-PCR, qRT-PCR that can show sufficient identification and
tissue-specific source for relevant body fluids will resolves a lot of forensic case analysis.
Therefore, RNA (mRNA and miRNA) successful amplification and analysis is optimistic tool
for future application in real case work to analysed more informative tissue specification
through molecular markers for LCN, old evidence, trace DNA, mixed DNA etc.
Word Count: 1965
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