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Analysis of genetic markers can provide clues for case investigation. Short tandem repeat (STR) detection and analysis are widely used for both personal identification and parentage testing. However, DNA analysis currently cannot provide sufficient information for body fluid identification. Tissue or cell sources of samples can be identified by detecting body fluid-specific mRNA markers, which have been studied thoroughly. Integrating STR profiling and mRNA expression patterns can provide more information than conventional methods for investigations and the reconstruction of crime scenes; this can be achieved by DNA/RNA co-extraction technology, which is economical, efficient, and suitable for low-template samples. Here, we propose a co-analysis system based on the PowerPlex 16 kit. This system can simultaneously amplify 25 markers, including 15 STRs, one non-STR amelogenin, and nine mRNA markers (three blood-specific, two saliva-specific, two semen-specific, and two housekeeping gene markers). The specificity and sensitivity of the co-analysis system were determined and aged and degraded samples were used to validate the stability of the co-analysis system. Finally, different DNA/RNA ratios and various carriers were evaluated. The results showed that the DNA/RNA co-analysis system correctly identified different types of body fluid stains. The STR profiles obtained using the co-analysis system were identical to those obtained using the PP16 kit, which demonstrates that the mRNA primers used did not affect STR profiling. Complete STR and mRNA profiles could be obtained from 1/8 portions of buccal swabs, 1/16 portions of swabs of blood and semen samples, 0.1 cm 2 of blood samples, 0.25 cm 2 of semen samples, and 1.0 cm 2 saliva samples. Additionally, our findings indicate that complete STR and mRNA profiles can be obtained with this system from blood and semen samples when the DNA/RNA ratio is 1:1/32. This study suggests that the co-analysis system could be used for simultaneous personal identification and body fluid identification., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Several studies have confirmed that microRNAs (miRNAs) are promising markers for body fluid identification since they were introduced to this field. However, there is no consensus on the choice of reference genes and identification strategies. In this study, 13 potential candidate miRNAs were screened from three forensically relevant body fluid datasets, and the expression of 12 markers in five body fluids was determined using a real-time quantitative method. Two probabilistic approaches, Naive Bayes (NB) and partial least squares discriminant analysis (PLS-DA), were then applied to predict the origin of the samples to determine whether probabilistic methods are helpful in body fluid identification using miRNA quantitative data. Furthermore, 14 reference combinations were used to validate the influence of different reference choices on the predicted results simultaneously. Our results showed that in the NB model, leave-one-out cross-validation (LOOCV) achieved 100% accuracy and the prediction accuracy of the test set was 100% in most reference combinations. In the PLS-DA model, the first two components could interpret about 80% expression variance and LOOCV achieved 100% accuracy when miR-92a-3p was used as the reference. This study preliminarily proved that probabilistic approaches hold huge potential in miRNA-based body fluid identification, and the choice of references influences the prediction results to a certain extent., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Non-protein coding RNA, miRNAs (microRNAs), are a class of promising molecular biomarkers for forensic body fluid identification (BFI) as their small size and tissue-specific expression manners. A number of studies have shown that semen can be distinguished from forensic-related body fluids (such as menstrual blood, venous blood, vaginal fluid, saliva, etc.) using semen-specific miRNAs through microassay screening and RT-qRCR. Infertility is becoming a global health problem, affecting 10%-15% of couples worldwide, and half of the cases are the result of male factors (Lian et al., 2009 [ 1 ]). Forensic researchers have to consider the impact of semen infertility on semen identification with a high incidence of infertility. In the present study, normal semen (NS) and four other types of infertile semen samples, including asthenospermia (AS), oligospermia (OS), azoospermia (AZ), oligospermia and asthenospermia (OSAS) semen, were collected. The expression levels of a set of semen-specific miRNA markers (miR-10a, miR-10b, miR-135a, miR-135b, miR-888 and miR-891a) were evaluated using a real-time quantitative PCR technique with a specific fluorescence-labelled TaqMan probe. The results showed the significantly high expression of these miRNAs in normal semen, and the molecules have semen specificity. Nevertheless, a distinct down-regulation in the expression of infertile samples compared with normal semen samples was observed. Moreover, differences in the results of selected optimal biomarkers between the discriminant function and two-dimensional scatter plots were also detected. The goal of the present study was to identify a small set of semen-specific miRNAs that efficiently and accurately distinguish semen (fertile and infertile) from other forensic-related body fluids. The results of the present study suggest that attention should be paid to infertile semen samples when using miRNAs to identify semen samples, for which would have a far-reaching impact on forensic identification. [ABSTRACT FROM AUTHOR]

• ARMS-PCR technique could effectively detect the methylation levels in a simple and cost-effective manner. • 22 body fluid-specific CpG sites were included in development of the multiplex assays. • The random forest model performed well in predicting single source body fluids with high prediction accuracy (99.66%). • Random forest model is a practical and promising prediction method for the mixed body fluid identification. A set of DNA methylation markers was detected and evaluated to identify body fluids using the amplification refractory mutation system-PCR (ARMS-PCR) and random forest algorithm. In this study, four multiplex DNA methylation reactions composed of 22 promising methylation markers were used to identify regular forensic body fluids, including venous blood, saliva, semen, menstrual blood, and vaginal fluid. The ARMS-specific primers were used to amplify the candidate markers, and then the methylation values of each CpG site were detected through capillary electrophoresis (CE). The DNA methylation patterns of 22 highly informative methylation markers were consistent with previously reported results to a certain extent. To our knowledge, our study is a new method to apply the ARMS-PCR technique and random forest model to detect DNA methylation patterns and identify the type of body fluids in forensic science, thus providing a new method for forensic body fluid identification. Moreover, we proved that this method is robust, applicable and effective for identifying body fluids using the random forest model. The accuracy to predict all body fluids reached up to 0.9966. We firmly believe that this method will have a great potential in the detection of methylation profiles at the molecular level. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have been of interest in forensic science for body fluid identification with recent years. However, there is no study investigating the species specificity of miRNA markers by the SYBR Green method. Due to the conservation of miRNAs across species, miRNA markers maybe less species-specific than mRNA markers, and in forensic cases, animal buccal swabs are more likely to appear. Therefore, in this study we addressed the influence of 8 kinds of animal buccal swabs on human saliva, semen, vaginal secretion swabs and blood identification with 10 candidate specific miRNA markers by the SYBR Green quantitative PCR. Our data showed that the expression levels of the candidate specific miRNA markers miR-124a and 372 in the cat, dog, mouse and rabbit buccal swabs were in the same range as the human vaginal secretion swabs; buccal swabs from these animals also showed similar expression levels to human saliva for the candidate specific miRNA markers miR-200c, 205 and 658. These results indicated that biomaterials of buccal swabs from cats, dogs, mice and rabbits may be mistaken for human saliva or human vaginal secretion swabs, both of which could result in false positives for human body fluids. Thus, the interpretation of these miRNA profiles for human body fluid identification can be inaccurate in the presence of these animal buccal swabs. Therefore, we suggested performing species tests before human body identification with miRNA markers. [ABSTRACT FROM AUTHOR]