RNA Analysis in Forensic Molecular Biology

Background: Different types of RNA take on multiple crucial functions in living cells and tissues. Messenger RNA (mRNA) is a temporary molecular carrier of genetic information. Analysis of the composition of all mRNA contained in a cell at a given moment, the so-called transcriptome, enables the determination of the type of cell and its condition, e.g., in pathologically altered states.

Method: This review is based on pertinent publications retrieved by a selective literature search.

Results: The analysis of differential gene expression has already been used in forensic molecular biology to determine the type of tissue contained in biological specimens. It is also being used in criminal investigations to determine the composition of mixed traces of various bodily fluids and/or organ tissues. The method is limited by degradation of the mRNA molecules through environmental influences. The use of newly developed molecular biological methods such as massive parallel sequencing can expand the information obtainable by this investigative method. Current research also addresses the forensic potential of deriving relevant information about the crime—e.g., its timing, or the condition of the involved persons—from the totality of mRNA species present in the specimens.

Conclusion: Forensic RNA analysis can yield a great deal of relevant information. It is likely to be applicable in a much wider variety of forensic situations in the near future.

Cite this as:

Courts C, Gosch A, Rothschild M: RNA analysis in forensic molecular biology. Dtsch Arztebl Int 2024; 121: 363–9 DOI: 10.3238/arztebl.m2024.0051

Forensic molecular biology can make decisive contributions towards solving the most serious of crimes, such as murder and sexual offenses, and for many years has been an indispensable part of forensic medicine and applied forensic sciences. Routine work of institutes of forensic medicine in Germany involves, amongst other things, molecular biological tests and the biostatistical evaluation of trace evidence and trace patterns, conducted in the course of investigations into various crimes. The identification of unknown deceased persons by DNA profiling as well as genetic parentage and family relationship analyses are also part of the typical routine tasks of German institutes of forensic medicine. Since the development of forensic DNA analysis with the detection of short tandem repeats (STR) at the beginning of the nineties, these highly objective forensic molecular biological tests are among the most important tools used in many criminal investigations. STR, also referred to as microsatellites, are short, usually non-coding DNA sequences of two to six base pairs in length, which are often found repeated in the genome of an organism, and allow the generation of individualized DNA profiles, even from minimal traces, which can be used in court. The reliable matching of biological trace evidence, such as blood stains, suspected traces of body fluids, hair, or skin epithelial cells, to a specific individual (also known as individualization) is therefore a crucial task in the field of forensic molecular genetics.

Methods of individualization have steadily improved, becoming more sensitive and more robust. It has long been possible to generate DNA profiles even from single cells (1) and to successfully analyze even severely degraded and aged material, for example, bones which have been submersed in water for decades or severely decomposed tissue (2, 3). Modern biostatistical methods, based on elaborate algorithms and simulations that can include significantly more data than established methods, now also allow calculations using even complex and incomplete DNA mixed profiles extracted from small amounts of degraded material to assess possible joint contribution to a crime by trace donors (4).

Change of perspective in forensic molecular biology

Over recent years, however, a change of perspective has occurred in forensic molecular biology and in the interpretation of its findings in court, with a noticeable shift of interest from the individualization to the contextualization of traces and trace patterns.

Contextualization is understood as the assignment of trace evidence according to its context of origin by drawing conclusions from purely material evidence, i.e. trace material, about certain, as well as quite different, aspects of a crime that led to the deposition of one or several traces or to the creation of the trace pattern or part of it. For this purpose, modern forensic molecular biology now uses several different contextualizing methods of investigation. This includes forensic DNA phenotyping (i.e., the deduction an individual’s externally visible characteristics from DNA) (5), molecular ballistics (6) (the molecular biological analysis of biological traces and trace patterns that are generated by gunshots directed at humans), and forensic RNA analysis (7).

Forensic RNA analysis

The first mention in the forensic literature of RNA extracted from postmortem tissue is to be found in a German-language article by Oehmichen et al. from 1984 on the postmortem biosynthesis of DNA and RNA (8). Since then, interest in mRNA analysis as well as in its forensic application options has significantly increased, especially in the processing of various contextualizing issues. Jawad et al. and Li et al. have presented current reviews on the potential of applying forensic RNA analysis (9, 10).

RNA also proved to be significantly more durable and less susceptible to degradation in postmortem tissues as well as in biological stains that were even decades old than previously assumed (11, 12). Further matrices for RNA extraction are constantly being developed. That said, postmortem degradation and the sensitivity of mRNA (Box) in particular to ubiquitous RNases remains a fundamental problem for its evaluation, requiring special precautions when collecting, storing, and processing trace evidence.

Box

Forensically relevant RNA species

Forensic RNA analysis for the identification of the type of trace material

The majority of biological stains used in forensic casework comprises, or originates from, skin contacts and human body fluids. The most common forensically relevant body fluids identified as the source of trace material are blood, saliva, semen, vaginal secretion, and menstrual blood. The first study results on the identification of body fluids using RNA expression were presented by Bauer and Patzelt in 2002 and 2003 (13, 14). Since then, numerous other relevant studies have been published which report on the RNA-based detection and identification of biological traces (overview at Sijen et al. [15]). This allows the detection or identification of one or more of the aforementioned, as well as other, biological traces, for example nasal secretions, sweat, or various organs.

The principle of RNA-based determination of trace type consists of detecting trace-specific RNAs in biological trace material, i.e., RNAs that are transcribed exclusively, or at least very commonly, in the cells of a certain trace type and so quantitively determine the presence of this type of trace evidence. The advantages of this technique over traditional and usually (immuno)chemical methods of identifying a trace type are significantly higher specificity, higher sensitivity, and the opportunity of successfully investigating even complex mixtures of trace elements. In addition, the RNA-based technique enables the precise identification of bodily secretions, such as vaginal or rectal secretions, for which no assays were previously available.

Use of a combined extraction technique also allows genomic DNA to be extracted simultaneously with RNA. This opens up the possibility of not only determining the type of trace material from even the smallest crime scene evidence, but also of creating the DNA profile of the person who deposited the trace, thus individualizing the evidence without further additional consumption of irreplaceable crime-scene samples (Figure 1).

Figure 1

Flowchart of the co-analysis of DNA and RNA in forensic casework

However, not all RNA markers presented so far for the identification of trace types are entirely specific. For example, commonly used markers for detecting vaginal secretion are also found in rectal mucosa, so the additional use of rectal mucosa-specific markers is required to be able to differentiate these traces (16). Nonspecific co-expression in other body fluids has also been observed in isolated cases for other markers. However, specificities of up to 100 percent can be achieved by combining several markers per body fluid and applying suitable interpretation guidelines (17, 18).

In a forensic setting, the identification of RNA is always possible in biological material where DNA is also identifiable, so that the RNA-based determination of body fluids can be applied even in a setting involving minimal trace amounts (a few microliters, or even less), as is often encountered in forensic casework (19).

The detection limit, i.e., the minimum quantity of RNA or biological trace material which must be available for the successful determination of body fluids, depends on a number of factors. These include, for example, the type of body fluid examined, the analyzed markers, the assay used, and inter-individual differences in expression. Due to the susceptibility of the single-stranded RNA molecules to enzymatic degradation by RNases present in the environment, the detectability of RNA may be reduced in comparison with DNA in damaged, aged, or inappropriately stored trace material. Given favorable storage conditions (room temperature, daylight, dryness), it is still possible to successfully undertake mRNA-based identification of body fluid after periods of years or even decades (20, 21). On the other hand, the survival capability of body fluid-specific mRNA-markers can be reduced to weeks or days under unfavorable conditions (high humidity, UV radiation, microbial growth) (21). Due to these limitations in mRNA expression analysis, the significantly shorter and more robust smallRNAs (miRNA or piRNA) with lengths of between 22 and 31 nucleotides (nt) can be considered relevant alternatives for forensic molecular biology investigations and analyzed in addition (22) (Box). However, given that smallRNAs are less well characterized in comparison with mRNAs, sometimes have lower tissue specificity, and also pose a greater analytical challenge due to the shortness of their molecules, the majority of currently used techniques for the RNA-based identification of body fluids are based on the recognition of differentially transcribed mRNA. Initial work on the combination of both RNA types in one assay has already been undertaken (22).

Another promising approach to improve the RNA-based identification of body fluids from old or degraded trace material is the examination of circular transcripts (Box) (23).

Published markers of various types of RNA for the identification of forensically relevant body fluids are summarized in the eTable.

eTable

Exemplary list of RNA markers used in published assays for the identification of forensically relevant body fluids

RNA-based identification of trace type for routine forensic use

Today, RNA-based identification of body fluids and organ tissues is so robust and mature that in some countries, including Germany since 2018, it is being used in routine forensic casework involving molecular biology. A number of specialized laboratories are accredited in accordance with DIN EN ISO/IEC 17025. These analyses are recognized by German courts and have already made an essential contribution towards ascertaining the truth in a number of cases where individualization of the source of evidence using DNA findings alone was not sufficient.

When doctors are involved in securing evidence during forensic examinations, for example of injured persons after a sexual offense, they should wear suitable protective clothing to avoid contamination and use certified RNase-free and self-drying forensic swabs. Once collected, specimens should then be stored in a dry and dark place at room temperature.

Detection methods in forensic RNA analysis

The detection of RNA markers specifically expressed in a certain cell type is usually undertaken using reverse transcriptase endpoint polymerase chain reaction (RT endpoint PCR). RNA is converted into complementary DNA (cDNA) and amplified, after which the enriched fragments are separated by length using capillary electrophoresis and detected by laser-induced fluorescence. The use of multiplex PCR assays allows around 20 to 30 transcripts to be examined simultaneously during one analysis. However, the procedure can only distinguish between the presence or absence of transcripts—quantitative recordings of transcript amounts are not possible.

Alternatively, the detection as well as a much more precise quantification of specific transcripts can also be achieved by using quantitative PCR (also known as real-time PCR). However, low multiplex capacity (four to five markers in one assay) and the associated increased material use limit its applicability in the field of forensics. Since 2015, innovative techniques for massively parallel sequencing (MPS, also commonly known as next generation sequencing [NGS]) of nucleic acid samples with high turnover have also been used in forensic RNA analysis (Haas et al. [24] provide an overview).

After their introduction in the mid-2000s (25), MPS techniques soon found their way into the forensic field, where their suitability for generating forensic DNA profiles was initially researched. Apart from its higher multiplex capacity, which allows it to analyze considerably more markers simultaneously from the same material, another advantage of the technique is that it not only determines the length of the respective fragments but can also read their nucleic acid sequence. Given that RNA molecules represent transcripts of DNA, their base sequence corresponds to the sequence of the DNA template. Typing and comparing characteristic genetic variants, so-called single nucleotide polymorphisms (SNPs), in the DNA and in corresponding RNA molecules from trace material make it possible to assign the components of a trace mixture to donors potentially involved in the crime (26). Until now, it was not possible using the traditional forensic RNA analysis methods to assign, for example, the components of a mixed trace of blood and saliva by means of DNA analysis specifically to individuals who had already been confirmed as contributors to the mixture (Figure 2). A first international laboratory comparison has already demonstrated the potential of this application (27).

Figure 2

Schematic representation of the RNA sequence-based assignment of donors to the components of a mixed trace

In the future, the multiplex capacity of MPS technology will also make it possible to combine RNA markers for several different contextualizing aspects in one assay. Thus, for example, it could be possible to determine in a single assay trace age in addition to identification of the trace type. The use of MPS in forensic RNA analysis can therefore contribute towards making the considerable amount of contextual information contained in the transcriptome accessible for forensic purposes.

Development potential and research projects for forensic RNA analysis

Apart from differences in transcriptome composition, some of which are quite pronounced due to physiological conditions, gene expression in cell populations of human body fluids and organ tissue are also regulated by other internal and external factors. There are a number of promising research projects dealing with the potential of the information contained in the transcriptome in answering forensically relevant issues.

Investigation of altered gene expression resulting from intrinsic processes

Many intrinsic processes are involved in modulating gene expression. Initial forensic projects are working, for example, on the development of methods for determining time since deposition of biological traces from gene expression, which can vary with the time of day (28, 29).

In addition, the diagnosis of certain clinical disorders, as distinct from non-natural causes of death, plays a decisive role in traditional forensic medicine. Particular difficulties are presented by sudden unexplained deaths (SUD), which occur without prior warning symptoms, have no morphological correlates, and are often due to previously unknown conditions of the heart.

Several studies have shown that certain cardiac disorders are associated with an altered expression of various mRNAs or other types of RNA (10, 24).

In future, the detection of such altered gene expression could therefore support forensic determination of the cause of death (molecular autopsy).

Investigation of altered gene expression resulting from extrinsic processes

Extrinsic factors can also bring about a change or adaptation in gene expression as a more or less systematic reaction. After an injury, for example from a knife wound, the wound-healing process begins immediately in vital tissue. Wound healing proceeds in chronologically ordered process phases (hemostasis, inflammation, proliferation, and matrix-remodeling). During these process phases, first platelets, then various types of immune cells, and later keratinocytes, fibroblasts, and endothelial cells are activated one after the other to promote the process of wound healing (30). Each wound-healing phase thus has a specific biochemical signature with its transcriptional correlate. While histological and immunohistochemical examinations are still widely used to deduce the age of a wound from stained tissue sections, more recent studies show that the examination of the transcriptome alone or in addition to the examination of the proteome can provide information about the age of a wound (31).

Furthermore, the ingestion of certain substances can trigger a reactive modulation of gene expression in various tissues. Initial studies on the possibility of detecting the ingestion of toxicologically relevant substances from changes in gene expression are already available (32, 33).

The analysis of transcript integrity and differential RNA degradation

Apart from alterations of gene expression itself, the completeness, or integrity, of RNA transcripts can provide information about forensically relevant issues. For example, examination of the transcriptome composition of biological traces which had been stored under different conditions for various periods of time showed that RNA molecules are degraded in these body fluids over time (34). The differential rate of degradation correlates with the age of the biological trace material (35). The degradation of nucleic acids in cells and tissues, which begins after the death of the human organism, also forms the basis for using transcript integrity to estimate post-mortem interval (PMI) (36).

These and other examples summarized in Figure 3 show that a variety of information is contained in the transcriptome which can be useful in many ways for forensic casework.

Figure 3

Versatility of forensic RNA analysis

Future prospects

While fundamental research has proven the multidimensionality and forensic relevance of the information content of the transcriptome, extensive validation efforts are still required in many areas. This includes the need to identify the best RNA markers, to develop mathematical models from the observed correlations, and to investigate the robustness of already established models under various forensically relevant conditions. A particular challenge here is the gathering of sufficiently large and informative sample sets, which, for example in the case of collecting specimens to estimate the age of the wound, often cannot be obtained systematically and must therefore be collected over long periods of time, taking into account many possible, non-standardizable influencing factors (such as the cause of death or the PMI) in the casework.

That said, when the constantly evolving technical possibilities, such as massively parallel sequencing, are considered, there are grounds for optimism that continued research efforts will make it possible in the future to use forensic RNA analysis to answer other forensic questions.

Conflict of interest statement
The authors declare that they have no conflicts of interests.

Manuscript received on 09 October 2023, revised version accepted on 06 March 2024

Translated from the original German by Dr. Grahame Larkin.

Corresponding author
Prof. Dr. Cornelius Courts
Forensic Medicine/Forensic Molecular Genetics

Forensic Molecular Genetics, 50823 Cologne

Cite this as:
Courts C, Gosch A, Rothschild M: RNA analysis in forensic molecular biology. Dtsch Arztebl Int 2024; 121: 363–9. DOI: 10.3238/arztebl.m2024.0051

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