Identification of Saliva Using MicroRNA Biomarkers for Forensic Purpose

In the forensic science community, microRNA (miRNA) profiling has started to be explored as an alternative tool for body fluid identification. Several origins of body fluid can be distinguished by measuring differential expression patterns of particular miRNAs. However, most of reported saliva miRNAs are nonoverlapping and debatable. The aim of this study was to develop a strategy of identifying saliva using miRNA biomarkers for forensic purpose. Eight miRNA candidates were selected to examine expression abundance in forensically relevant body fluids using hydrolysis probes quantitative real‐time PCR (TaqMan qPCR). Results revealed that none of them was truly saliva specific, and only miR‐200c‐3p, miR‐203a, and miR‐205‐5p were higher or more moderate expression in saliva. A stepwise strategy that combines each of three miRNAs with different body fluid‐specific miRNAs was developed, and three miRNA combinations could effectively differentiate saliva from other body fluids.     

Multiplex high resolution melt (HRM) messenger RNA profiling assays for body fluid identification

Traditional body fluid identification methods use a variety of technologically diverse techniques that do not permit the identification of all body fluids. Definitive identification of the biological material present can be crucial to a fuller understanding of the circumstances pertaining to a crime. Thus definitive molecular based strategies for the conclusive identification of forensically relevant biological fluids need to be developed. Messenger (mRNA) profiling is an example of such a molecular based approach.Current mRNA body fluid identification assays typically involve either capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the time required for analysis. For qRT-PCR assays, only 3 or 4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays and to reduce the time and cost of analysis, we have developed multiplex high resolution melt (HRM) assays that provide an identification of all forensically relevant biological fluids and tissues.     

Binary logistic regression models enable miRNA profiling to provide accurate identification of forensically relevant body fluids and tissues

Numerous studies have demonstrated the ability to identify the body fluid of origin of forensic biological stains using messenger (mRNA) profiling. However, the size of the amplification product used in these assays (100–400 bases) may not be ideal for use with environmentally degraded samples. MiRNA profiling represents a potential alternative to mRNA profiling, since the small size of the miRNAs (∼22 bases) might still permit their detection in degraded stains. Previously, we reported the first study involving the forensic use of microRNA (miRNA) profiling, which required screening of 452 candidates. Since our initial screening, hundreds of novel miRNAs have been identified. We have therefore evaluated additional miRNA candidates to further improve the sensitivity and specificity of the body fluid assays. Consequently we have expanded our body fluid identification panel to include 18 miRNAs (comprising 5 original and 13 novel miRNAs). This panel permits the identification of all forensically relevant body fluids and, uniquely, includes miRNAs for the identification of skin.Using normalized miRNA expression data, we constructed body fluid specific binary logistic regression models to permit an accurate identification of the body fluid of interest. Using the developed models, we have obtained 100% accuracy in predicting the body fluid of interest.     

Development and validation of molecular methodologies for identification of biological traces from crime scenes

This study aimed to develop a methodology to identify biological fluids in sexual assault cases through mRNA markers. Biological fluid samples such as blood, saliva, and semen were collected from volunteers and submitted to RT-qPCR reactions with specific primers for the biomarkers HTN3 (saliva), ALAS (blood) andTGM4 (semen). The Melting (Tm) of each biological fluid was analyzed and the result inferred a high specificity capable of differentiating such traces. Biplex systems were generated to improve trace analysis in a single qPCR reaction.     

Evidence based strategy for normalization of quantitative PCR data,in forensic miRNA-analysis

Micro-RNA (miRNA) based analysis of body fluids and composition of complex crime stains has recently been introduced as a potential and powerful tool to forensic genetics. Analysis of miRNA analysis has several advantages over mRNA but reliable miRNA detection and quantification using quantitative PCR requires a solid and forensically relevant normalization strategy. In our study we evaluated a panel of 12 carefully selected reference genes for their suitability as endogenous controls in miRNA qPCR normalization in forensically relevant settings. We analyzed assay performances and variances in venous blood, semen, menstrual blood, saliva and vaginal secretion and mixtures thereof integrating highly standardized protocols with contemporary methodologies and included several well established computational algorithms.Based on these empirical results, we recommend normalization to the group of RNU24, RNU43, and RNU66, as this signature exhibits the most stable expression levels and the least expected variation among the evaluated candidate reference genes in forensically relevant body fluids. To account for the lack of consensus on how best to perform and interpret quantitative PCR experiments, our study's documentation is according to MIQE guidelines, defining the “minimum information for publication of quantitative real-time PCR experiments”.     

Messenger RNA Profiling for Forensic Body Fluid Identification： Research and Applications

Identifying the origin of body fluids left at a crime scene can give a significant insight into crime scene reconstruction by supporting a link betw een sample donors and actual criminal acts. How ev-er, the conventional body fluid identification methods are prone to various limitations, such as time con-sumption, intensive labor, nonparallel manner, varying degrees of sensitivity and limited specificity. Re-cently, the analysis of cell-specific messenger RNA expression （mRNA profiling） has been proposed to supplant conventional methods for body fluid identification. Since 2011, the collaborative exercises have been organized by the European DNA Profiling Group （EDNAP ） in order to evaluate the robustness and reproducibility of mRNA profiling for body fluid identification. The major advantages of mRNA profil-ing, compared to the conventional methods, include higher sensitivity, greater specificity, the ability of detecting several body fluids in one multiplex reaction, and compatibilitywith current DNA extraction and analysis procedure. In the current review ,we provided an overview of the present know ledge and detection methodologies of mRNA profiling for forensic body fluid identification and discussed its possi-ble practical application to forensic casew ork.     

Informativeness of NGS Analysis for Vaginal Fluid Identification

The identification of vaginal fluids in forensic examinations plays an important role in crime scene reconstruction. Molecular detection of vaginal bacterial communities can lead to the correct discrimination of body fluids. These kinds of studies can be performed through multiplex real‐time PCR using primers for a specific selection of bacteria. The availability of next‐generation sequencing (NGS) protocols provided for the extension of the analysis to evaluate the prokaryotes present in specimens. In this study, DNA was extracted from 18 samples (vaginal, oral, fecal, yoghurt) and analyzed by real‐time PCR and NGS. The comparison between the two approaches has demonstrated that the information developed through NGS can augment the more conventional real‐time PCR detection of a few key bacterial species to provide a more probative result and the correct identification of vaginal fluid from samples that are more forensically challenged.     

Background Levels of Salivary-α-amylase Plus Foreign DNA in Cases of Oral Intercourse: a Female Perspective

Saliva plus DNA from a suspect is commonly encountered in sexual assault cases on bodily swabs. However, without background knowledge, the weight of this evidence is unknown. It may indicate the presence of saliva resulting from cunnilingus, or it may represent indirect transfer. In this study, females who refrained from cunnilingus donated 43 items of underwear and 19 vaginal swabs. The samples were subjected to Phadebas^®, RSID^™-Saliva and mRNA profiling and were subsequently DNA-profiled to determine the prevalence of background saliva in the female population. The results report that 15.8% of females who refrained from cunnilingus were positive for saliva and a further 10.5% also had DNA from unknown source(s). These findings of the rate of indirect transfer were evaluated with the Bayesian approach, and it was found that the evidence of saliva plus a high foreign DNA source adds moderately strong support to the allegation of cunnilingus.     

A 1-year time course study of human RNA degradation in body fluids under dry and humid environmental conditions

Blood, saliva and semen are some of the forensically most relevant biological stains found at crime scenes. mRNA profiling is a reliable approach for the identification of the origin of an evidentiary trace. A stable set of markers and the knowledge about the effects of RNA degradation under different environmental conditions is necessary for the determination of an unknown biological stain. The aim of this work was to compare RNA degradation for human blood, semen and saliva at three different concentrations during a 1-year time period and exposed to dry and humid conditions. Also, this study addressed the question whether there are relevant differences in the efficiency of two RNA extraction methods.     

Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood.     

Raman spectroscopy offers great potential for the nondestructive confirmatory identification of body fluids

Raman spectroscopy was used to compare body fluids commonly found at crime scenes in a nondestructive manner. The dry traces of semen, vaginal fluid, sweat, saliva, and blood were analyzed using confocal Raman microscopy with a 785-nm excitation. The results show that the five fluids can be differentiated from one another by visual comparison of their Raman spectra, and that the laser radiation does not damage the sample. The Raman signature of each body fluid is specific and correlates with the known composition of the fluid. Dry traces of human and canine semen exhibited distinctly different Raman signatures. Overall, this preliminary study demonstrates the great potential of Raman spectroscopy for nondestructive, confirmatory identification of body fluids for forensic purposes.