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. METHODS: 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.

Forensically relevant anatomical brain regions cannot be sub-differentiated by RNA expression analysis.

The contextualization of biological traces generated by severe head injuries can be beneficial for criminal investigations. Here we aimed to identify and validate mRNA candidates for a robust sub-differentiation of forensically and traumatologically relevant brain regions. To this purpose, massively parallel sequencing of whole transcriptomes in sample material taken from four different areas of the cerebral cortex (frontal, temporal, parietal, occipital lobe) was performed, followed by bioinformatical data analysis, classification, and biostatistical candidate selection. Candidates were evaluated by Multiplex-RT-PCR and capillary electrophoresis. Only a weak relative upregulation and solely for candidates expressed in the parietal lobe was observed. Two candidates with upregulation in the cerebellar region (PVALB and CDR2L) were chosen for further investigation; however, PVALB could not reliably and repeatedly be detected in any lobe whereas CDR2L was detectable in all lobes. Consequently, we suggest that differences in mRNA expression between four regions of the cerebral cortex are too small and less pronounced to be useful for and applicable in forensic RNA analysis. We conclude that sub-differentiation of these brain regions via RNA expression analysis is generally not feasible within a forensic scope.

Spitting in the wind?-The challenges of RNA sequencing for biomarker discovery from saliva.

Forensic trace contextualization, i.e., assessing information beyond who deposited a biological stain, has become an issue of great and steadily growing importance in forensic genetic casework and research. The human transcriptome encodes a wide variety of information and thus has received increasing interest for the identification of biomarkers for different aspects of forensic trace contextualization over the past years. Massively parallel sequencing of reverse-transcribed RNA ("RNA sequencing") has emerged as the gold standard technology to characterize the transcriptome in its entirety and identify RNA markers showing significant expression differences not only between different forensically relevant body fluids but also within a single body fluid between forensically relevant conditions of interest. Here, we analyze the quality and composition of four RNA sequencing datasets (whole transcriptome as well as miRNA sequencing) from two different research projects (the RNAgE project and the TrACES project), aiming at identifying contextualizing forensic biomarker from the forensically relevant body fluid saliva. We describe and characterize challenges of RNA sequencing of saliva samples arising from the presence of oral bacteria, the heterogeneity of sample composition, and the confounding factor of degradation. Based on these observations, we formulate recommendations that might help to improve RNA biomarker discovery from the challenging but forensically relevant body fluid saliva.

Selecting mRNA markers in blood for age estimation of the donor of a biological stain.

RNA has gained a substantial amount of attention within the forensic field over the last decade. There is evidence that RNAs are differentially expressed with biological age. Since RNA can be co-extracted with DNA from the same piece of evidence, RNA-based analysis appears as a promising molecular alternative for predicting the biological age and hence inferring the chronological age of a person. Using RNA-Seq data we searched for markers in blood potentially associated with age. We used our own RNA-Seq data from dried blood stains as well as publicly available RNA-Seq data from whole blood, and compared two different approaches to select candidate markers. The first approach focused on individual gene analysis with DESeq2 to select the genes most correlated with age, while the second approach employed lasso regression to select a set of genes for optimal prediction of age. We present two lists with 270 candidate markers, one for each approach.

Nothing but hot air?-On the molecular ballistic analysis of backspatter generated by and the hazard potential of blank guns.

Blank cartridge guns are prevalent especially in countries with laws restricting access to conventional firearms, and it is a common misconception that these weapons are harmless and only used as toys or for intimidation. However, although their harming potential is well-documented by numerous reports of accidents, suicides, and homicides, a systematic molecular biological investigation of traces generated by shots from blank cartridges at biological targets has not been done so far. Herein, we investigate the occurrence and analyzability of backspatter generated by shots of different types of blank cartridge guns firing different types of blank ammunition at ballistic gelatin model cubes doped with human blood and radiological contrast agent soaked into a spongious matrix and covered with three different variants of skin simulants. All skin simulants were penetrated, and backspatter was created in 100% of the shots in amounts sufficient for forensic short tandem repeat (STR) typing that resulted in the correct identification of the respective blood donor. Visible backspatter was documented on the muzzle and/or inside the barrel in all cases, and in 75% of cases also on the outer surfaces and on the shooter's hand(s). Wound cavities were measured and ranged between 1 and 4.5 cm in depth. Discussing our findings, we provide recommendations for finding, recovering, and analyzing trace material from blank guns, and we demonstrate the considerable hazard potential of these devices, which is further emphasized by the presentation of a comprehensive overview of the pertinent literature on injuries inflicted by blank guns.

Ten years of molecular ballistics-a review and a field guide.

Molecular ballistics combines molecular biological, forensic ballistic, and wound ballistic insights and approaches in the description, collection, objective investigation, and contextualization of the complex patterns of biological evidence that are generated by gunshots at biological targets. Setting out in 2010 with two seminal publications proving the principle that DNA from backspatter collected from inside surfaces of firearms can be retreived and successfully be analyzed, molecular ballistics covered a lot of ground until today. In this review, 10 years later, we begin with a comprehensive description and brief history of the field and lay out its intersections with other forensic disciplines like wound ballistics, forensic molecular biology, blood pattern analysis, and crime scene investigation. In an application guide section, we aim to raise consciousness to backspatter traces and the inside surfaces of firearms as sources of forensic evidence. Covering crime scene practical as well as forensic genetic aspects, we introduce operational requirements and lay out possible procedures, including forensic RNA analysis, when searching for, collecting, analyzing, and contextualizing such trace material. We discuss the intricacies and rationales of ballistic model building, employing different tissue, skin, and bone simulants and the advantages of the "triple-contrast" method in molecular ballistics and give advice on how to stage experimental shootings in molecular ballistic research. Finally, we take a look at future applications and prospects of molecular ballistics.

mRNA profiling of mock casework samples: Results of a FoRNAP collaborative exercise.

In recent years, forensic mRNA profiling has increasingly been used to identify the origin of human body fluids. By now, several laboratories have implemented mRNA profiling and also use it in criminal casework. In 2018 the FoRNAP (Forensic RNA Profiling) group was established among a number of these laboratories with the aim of sharing experiences, discussing optimization potential, identifying challenges and suggesting solutions with regards to mRNA profiling and casework. To compare mRNA profiling methods and results a collaborative exercise was organized within the FoRNAP group. Seven laboratories from four countries received 16 stains, comprising six pure body fluid / tissue stains and ten mock casework samples. The laboratories were asked to analyze the provided stains with their in-house method (PCR/CE or MPS) and markers of choice. Five laboratories used a DNA/RNA co-extraction strategy. Overall, up to 11 mRNA markers per body fluid were analyzed. We found that mRNA profiling using different extraction and analysis methods as well as different multiplexes can be applied to casework-like samples. In general, high input samples were typed with high accuracy by all laboratories, regardless of the method used. Irrespective of the analysis strategy, samples of low input or mixed stains were more challenging to analyze and interpret since, alike to DNA profiling, a higher number of markers dropped out and/or additional unexpected markers not consistent with the cell type in question were detected. It could be shown that a plethora of different but valid analysis and interpretation strategies exist and are successfully applied in the Forensic Genetics community. Nevertheless, efforts aiming at optimizing and harmonizing interpretation approaches in order to achieve a higher consistency between laboratories might be desirable in the future. The simultaneous extraction of DNA alongside RNA showed to be an effective approach to identify not only the body fluid present but also to identify the donor(s) of the stain. This allows investigators to gain valuable information about the origin of crime scene samples and the course of events in a crime case.

A distant relationship?-investigation of correlations between DNA isolated from backspatter traces recovered from firearms, wound profile characteristics, and shooting distance.

In molecular ballistics, where traces originating from the use of firearms against biological targets are investigated, "backspatter" traces are of particular importance. This biological material comprising blood and tissue from the victim is propelled back from the bullet entry site towards the direction of the shooter and can consolidate and persist on the inner and outer surfaces of the firearm, from where it can be collected and analyzed. Thus, a connection between the weapon and the victim can be established solely by molecular biological trace analysis. For the criminalistic investigation of gun-related crimes, the determination of the distance between the weapon and the victim can be of critical importance in reconstructing the circumstances of a crime. In this study, we investigated possible correlations between the shooting distance and the amount of backspatter in/on the used firearm. To this purpose, we employed a previously established skull model and performed shootings in triplicates from various distances up to 50 cm with two types of handguns (pistol and revolver). Backspatter was collected from various sampling locations, and DNA contents were quantified. A post-shooting wound channel evaluation was conducted by optical and radiological evaluation. The obtained DNA yields varied considerably between replicates from the same and from different distances. In contrast, apart from contact shots, no meaningful differences were observable in wound channel evaluations. In summary, no meaningful correlation between backspatter distribution and DNA yields, the shooting distance and the condition of the wound channel could be established.

DNA transfer to firearms in alternative realistic handling scenarios.

Firearms are the most relevant items of evidence in gun-related crimes, likely bearing various traces facilitating an objective reconstruction of the crime. Trace DNA recovered from firearm surfaces might help to identify individual(s) having handled the firearm and thereby possibly to link the firearm and the corresponding shooter, however, the interpretation of DNA traces on handled items can be challenging and requires a detailed understanding of various factors impacting DNA prevalence, transfer, persistence and recovery. Herein, we aimed at improving our understanding of factors affecting the variability of trace DNA characteristics recovered from firearms handled in gun-related crimes: Skin contact traces were recovered from various outer surfaces of two types of firearms handled in four realistic, casework-relevant handling scenarios and the corresponding trace characteristics (DNA yield, number of contributors, relative profile contribution for known and unknown contributors, LRs) were compared. Trace DNA characteristics differed distinctly between handling conditions, firearm and surface types as well as handling individuals and intraindividual deposits emphasizing the variability and complexity of trace DNA profile composition expected to be recovered from firearms after realistic handling scenarios. The obtained results can provide useful insights for forensic experts evaluating alternative activity level propositions in gun-related crimes.

Evaluation of the backspatter generation and wound profiles of an anatomically correct skull model for molecular ballistics.

Molecular ballistics connects the molecular genetic analysis of biological traces with the wounding events and complex forensic traces investigated in terminal ballistics. Backspatter, which originates from a projectile hitting a biological target when blood and/or tissue is propelled back into the direction of the gun, is of particular interest; those traces can consolidate and persist on the outer and inner surfaces of firearms and serve as evidence in criminal investigations. Herein, we are the first to present an anatomically correct head model for molecular ballistic research based on a polyurethane skull replica enclosing tissue-simulating sponge material that is doped with "triple-contrast" mixture (EDTA-blood, acrylic paint, and an x-ray contrast agent). Ten percent ballistic gelatin was used as brain simulant. We conducted contact and intermediate-range shots with a Glock 19 pistol (9 mm Luger), a pump-action shotgun (12/70 slugs), and blank cartridge handguns. Each shot was documented by a high-speed camera at 35,000 fps. Apart from the blank cartridge guns, all gunshots penetrated the skull model and created backspatter, which was recovered from the distal part of the barrels and analyzed. The pistol contact shots and one of three shotgun shots yielded full STR profiles. While the shotgun slugs destroyed the skulls, the remaining models could be used for radiological and optical fracture and wound channel evaluation. Known backspatter mechanisms and their respective timing could be confirmed visually by video analysis. Our complete model setup proved to be well applicable to molecular ballistic research as well as wound channel and fracture pattern investigation.

On DNA transfer: The lack and difficulty of systematic research and how to do it better.

Since DNA from touched items and surfaces ("touch DNA") can successfully and reliably be analyzed, the question as to how a particular DNA containing sample came to be from where it was recovered is of increasing forensic interest and expert witnesses in court are increasingly challenged to assess for instance whether an incriminatory DNA sample matching to a suspect could have been transferred to the crime scene in an innocent manner and to guess at the probability of such an occurrence. The latter however will frequently entail expressing a subjective probability i.e. simply making a best guess from experience. There is, to the present date, an extensive and complex body of literature on primary, secondary, tertiary and even higher order DNA transfer, its possibility, plausibility, dependency on an array of variables and factors and vast numbers of permutations thereof. However, from our point of view there is a lack of systematic data on DNA transfer with existing research widely varying in quality and relevance. Our aim was, starting from a comprehensive survey of the status quo and appreciating its increasing importance, to in the first part of our review raise consciousness towards the underestimated and insufficiently accounted for complexity of DNA transfer and thus appendant research of forensic scientists serving as expert witnesses in court but also acting in the role of a journal referee to point them to areas of criticism when reviewing a manuscript on DNA transfer. In the second part, we present propositions how to systematize and integrate future research efforts concerning DNA transfer. Also, we present a searchable database providing an extensive overview of the current state of knowledge on DNA transfer, intended to facilitate the identification of relevant studies adding knowledge to a specific question and thus help forensic experts to base their opinion on a broader, more complete and more reproducible selection of studies.

All mixed up?-genotype change after stem cell transplantation impeded verification of 21-year-old semen sample-a case report.

We report a case of identity testing in which a patient charged us with the verification of a semen sample that he had donated and cryopreserved more than 20 years ago and now was suspecting of having been inadvertently interchanged. We found a non-match of the DNA profiles of the patient's blood and the semen samples but could show that this was due to the patient having received a stem cell transplantation of his full brother as part of a cancer therapy in 1997 which was not known to us when the samples were first tested. Also, the blood and semen samples exhibited a low probability of full sibship at first supporting the patient's suspicion that his semen sample might indeed have been interchanged. By also testing Y-STRs and including hair roots in the DNA analysis, we could show that the transplant did indeed originate from the patient's brother and that the semen sample did indeed originate from the patient.

RNA/DNA co-analysis from bloodstains on aged polyvinyl-alcohol gloves prepared for securing evidence from the hands of victims of fatal gunshot injuries.

In contrast to cumulative techniques (e.g., tape-lift) for qualitative gunshot residues (GSR) analysis, topographic methods are commonly applied to preserve the integrity of evidence from a shooter's or victim's hand in cases of gun-related crimes. Topographic sampling techniques employing adhesive foils, latex, or the polyvinyl alcohol (PVAL) method enable unambiguous sampling of biological and non-biological trace material while preserving its spatial distribution and relation to each other. The PVAL method in particular allows for a topographically veridic and quantitative conservation of traces of GSR and biological stains that are embedded in the PVAL glove, because it completely removes these traces from the hand. The present study investigated the success rates of STR profiling and the detection of blood and brain-specific gene expression from minimal traces of blood splatter as well as parallel to the positive detection of gunshot residues embedded in 17 PVAL gloves taken from the hands of deceased persons in the context of homicide cases in the period between 1996 and 2003. The water-soluble PVAL matrix is shown to be fully compatible with successful STR profiling and the detection of blood- and brain-specific miRNA expression, even after up to 20 years of storage, demonstrating that this sampling technique offers advantages compared to other more simplistic sampling methods like taping.

Alterations in gene expression after gamma-hydroxybutyric acid intake-A pilot study.

Gamma-hydroxybutyric acid (GHB) acts as an agonist of the GABAB receptor, where GHB induces a depressant effect in the central nervous system. Besides its therapeutic application, GHB is also used as a date rape drug. However, the detection of GHB ingestion proves to be difficult due to its narrow detection window. The aim of this pilot study was to assess differential gene expressions after GHB intake to identify potential biomarkers for the detection of GHB intake. To this aim, alteration in gene expression of ALDH5A1, AKR7A2, EREG, and PEA15 was investigated via quantitative PCR (qPCR). Data normalization was based on a previously established and empirically derived normalization strategy. Blood samples of patients (n = 3) therapeutically taking sodium oxybate solution (GHB) and of donors without GHB intake (n = 49) were analyzed and compared. All qPCR procedures and results are reported according to the MIQE guidelines. Investigation of suitable reference genes using established algorithms suggested PPIB and FPGS as best-suited normalizers. Alterations in gene expression relating to GHB intake could not be confirmed to a forensically sufficient degree. However, significant differences in expression of EREG in the control group were observed, when time-point of sample collection was considered, indicating circadian rhythm. The study's main limitation is the small number of study subjects. Herein, we are first to present an empirically derived strategy for a robust normalization of qPCR data from the analysis of GHB-induced gene expression in human blood. We present results of the analysis of differential expression of ALDH5A1, AKR7A2, EREG, and PEA15 in the GHB-negative population. Finally, we report our findings on the effect of GHB intake on the expression of these genes and their presumable potential as GHB biomarkers.

RNA/DNA co-analysis on aged bloodstains from adhesive tapes used for gunshot residue collection from hands.

In cases of firearm related fatalities a systematic investigation at the scene of death is indispensable to differentiate between self-inflicted and homicidal gunshot injuries. A common method to preserve gunshot residues (GSR) is their collection using adhesive tapes. However, the biological material gathered at the same time by the tapes would be of special interest if backspatter, ejected from the entrance wound against the direction of fire, could be detected. In the present study we examined the success rate of co-analysis of RNA and DNA recovered from biological traces sampled with adhesive tapes. The material originated from eight cases of fatal gunshots, taken from the hands of suspects or victims, examined 5 to 19 years ago for GSR. For all types of adhesive tapes tested, quantity and quality of the co-extracted nucleic acids was insufficient for successful DNA profiling, but was sufficient for the detection of blood-specific micro RNA (miRNA). In summary, sampling trace evidence from the hands of persons involved in fatal gunshots with adhesive tapes has a long-term detrimental effect on biological traces.

Identification of organ tissue types and skin from forensic samples by microRNA expression analysis.

The identification of organ tissues in traces recovered from scenes and objects with regard to violent crimes involving serious injuries can be of considerable relevance in forensic investigations. Molecular genetic approaches are provably superior to histological and immunological assays in characterizing organ tissues, and micro-RNAs (miRNAs), due to their cell type specific expression patterns and stability against degradation, emerged as a promising molecular species for forensic analyses, with a range of tried and tested indicative markers. Thus, herein we present the first miRNA based approach for the forensic identification of organ tissues. Using quantitative PCR employing an empirically derived strategy for data normalization and unbiased statistical decision making, we assessed the differential expression of 15 preselected miRNAs in tissues of brain, kidney, lung, liver, heart muscle, skeletal muscle and skin. We show that not only can miRNA expression profiling be used to reliably differentiate between organ tissues but also that this method, which is compatible with and complementary to forensic DNA analysis, is applicable to realistic forensic samples e.g. mixtures, aged and degraded material as well as traces generated by mock stabbings and experimental shootings at ballistic models.