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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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Microhaplotype markers are emerging forensic genetic markers that have received broad attention in forensics and may supplement existing genetic marker panels. Short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs) are the general genetic markers at present. Stutter and the high mutation rate of STR markers and the low polymorphism of SNP markers obstruct the solving of certain cases. Kidd proposed microhaplotype markers that encompass 2-4 SNPs. In this study, we screened microhaplotype loci through three criteria, and chose the Illumina ® MiSeq platform to sequence the new markers. A new nomenclature was proposed and Perl-based tool FLfinder was designed to genotype the microhaplotype marker. After counting the number of haplotypes in samples that were sequenced and calculating common forensic parameters, 13 loci with high polymorphism were reported. Twelve of the 13 loci had an average allele coverage ratio (ACR) of 0.72 to 0.92. Structure analysis showed that 2504 samples (1000 genome project) could be divided into 5 groupings of populations, and each one representing a continental origin. The finding indicates that microhaplotype markers could be used for individual identification and ancestry inference, and a new choice is provided for forensic practice in the future., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The resolution of DNA mixtures is still a difficult problem that is worthy of further study. A common method applied for analysing mixtures is the use of autosomal STR markers as well as related calculation software based on genotypes; however, these markers have a limitation in detecting minor DNA in unbalanced mixtures if major DNA constitutes over 95% of the stain. Novel biomarkers, such as Y-STR, DIP-STR and SNP-STR, have been shown to perform well in distinguishing DNA donors in this type of mixture. DIP-STR can successfully target minor DNA in 1000-fold background DNA using two separate allele-specific primers. However, whether this method can successfully detect minor DNA primarily depends on the distribution of the DIPs in a population. Until now, only Swiss population data have been reported; therefore in this study, we selected 10 DIP-STR markers that performed well in the Swiss population and investigated whether these markers were also useful among the southwest Chinese Han population. The allele frequencies were estimated based on 152 samples, and six of the ten DIP-STR makers had a relatively high probability of informative markers (I value), which indicated their potential usefulness in the southwest Chinese Han population. A comparative study of DIP-STR markers and autosomal STR markers demonstrated that DIP-STR markers detected minor DNA at a ratio of 1:1000, while autosomal STR markers often failed to genotype minor DNA because of strong background noises caused by large amount of major DNA. However, the discrimination power was not high enough using these six DIPs alone. Therefore, we suggest that development of a panel with more loci is imperative and that a panel combined with DIP-STR and SNP-STR markers may be a possible way to achieve better discrimination power., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 cm2 of blood samples, 0.25 cm2 of semen samples, and 1.0 cm2 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. [ABSTRACT FROM AUTHOR]

Postmortem interval (PMI) determination is an important part of criminal investigations, but it is still subject to uncertainty. Degradation of mRNA in PMI determination has been studied in decays; however, some studies have reported no correlation between PMI and RNA degradation. Thus, we aimed to determine whether RNA quantity was correlated with PMI. Heart and brain tissues were separated from a mouse model of a 0–48 h PMI with 29 time points. We then coextracted the DNA and RNA in one tube with Bioteke coextraction kits and selected some mRNA markers associated with cell oxygen deprivation and apoptosis as target genes, such as hypoxia-associated factor (HAF), apoptosis-inducing factor (AIF), hypoxia-inducible factor 2 alpha (HIF2a), and factor inhibiting HIF (FIH). We measured the quantity of these markers using real-time quantitative PCR (qPCR), and Caspase-3 DNA and 18S were each used for normalization. The results showed that in the heart tissue, the degradation of HIF2a, AIF, and FIH was correlated with PMI, as was the degradation of HIF2a, FIH, and AIF in brain tissue when normalized with Caspase-3 DNA. However, when normalized with 18S, only the degradation of HIF2a in brain tissue was correlated with PMI. Interestingly, the quantity of HAF in brain tissue was found to increase after death with either 18S or Caspase-3 DNA normalization, and it was significantly correlated with 0–48 h PMI. These results indicated that mRNA quantity can be used to determine PMI and that Caspase-3 DNA is feasible for PMI estimation. In summary, we established mathematical models for PMI determination using multiple mRNA markers and multiple tissues and further studies are needed to validate and investigate these markers and mathematical models in human tissues. Duo Peng and Meili Lv contributed equally to this work. [ABSTRACT FROM AUTHOR]

Highlights • A novel nomenclature of microhaplotype marker is proposed. • A new Perl-based tool Forensic-Loci-finder (FLfinder) was designed to profile the microhaplotype markers. • The direct and accurate genotypes of microhaplotype markers were got using massively parallel sequencing (MPS). • 1000 Genomes Phase 3 data was used to give a point that microhaplotype markers have the potential of ancestry inference. Abstract Microhaplotype markers are emerging forensic genetic markers that have received broad attention in forensics and may supplement existing genetic marker panels. Short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs) are the general genetic markers at present. Stutter and the high mutation rate of STR markers and the low polymorphism of SNP markers obstruct the solving of certain cases. Kidd proposed microhaplotype markers that encompass 2–4 SNPs. In this study, we screened microhaplotype loci through three criteria, and chose the Illumina® MiSeq platform to sequence the new markers. A new nomenclature was proposed and Perl-based tool FLfinder was designed to genotype the microhaplotype marker. After counting the number of haplotypes in samples that were sequenced and calculating common forensic parameters, 13 loci with high polymorphism were reported. Twelve of the 13 loci had an average allele coverage ratio (ACR) of 0.72 to 0.92. Structure analysis showed that 2504 samples (1000 genome project) could be divided into 5 groupings of populations, and each one representing a continental origin. The finding indicates that microhaplotype markers could be used for individual identification and ancestry inference, and a new choice is provided for forensic practice in the future. [ABSTRACT FROM AUTHOR]

In the past decades, mRNA marker has been well demonstrated the remarkable progress and wide application for its high sensitivity, specificity and robustness. However, according to previous studies, infertility has become a global health problem. Therefore given the increasing occurrence of male infertility, forensic researchers have to consider the impact of semen infertility on semen identification. In present study, normal semen (NS) asthenospermia (AS) and oligospermia (OS) semen samples were collected. The expression of semen-specific mRNA markers (KLK3, PRM1) was detected using end-point PCR. The results showed that KLK3 was almost not influenced by infertile semen samples. In contrast, PRM1 couldn't be detected in all the infertile semen samples, indicating that it may not be ideal mRNA marker to identify semen samples. The present study aimed to explore the effect of the infertile semen on body fluid identification and to identify semen-specific mRNAs. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) has attracted wide interest of the forensic community since Hanson attempted to search specific microRNAs for body fluid identification from microarray. Research groups have screened out some sets of miRNA markers that can be used in forensic body fluids identification up to now. Most of those miRNA markers could not be repeated in different labs. The markers confirmed by SYBRgreen always could not be confirmed by another group in Taqman. Different reverse-transcription quantitative PCR methods might be the major cause of that. To seek out the influences of different reverse-transcription quantitative PCR methods on the expression of miRNA, we chose three miRNAs which showed conflicted or consistent results in different RT-qPCR methods according to previous studies, including miR-412-3p, miR-124a-3p, miR-10b-5p, for menstrual blood, vaginal secretions and semen respectively. MiRNAs were analyzed through Taqman and SYBRgreen according to the protocols respectively. Different ΔCq values of the same miRNA marker were detected by SYBRgreen and Taqman. Different results can be attributed to the sensitivity and specificity of two reverse-transcription quantitative methods. The choice of appropriate reverse-transcription quantitative method should be based on the study targets and the conditions of samples. [ABSTRACT FROM AUTHOR]

DIPs (insertion/deletion polymorphisms) combining the advantages of both SNPs (single nucleotide polymorphisms) and STRs (short tandem repeats) show a great value in forensic case works. A high sensitive 21 AIM (ancestry informative markers)-DIPs panel developed by Daniel et al. is sufficient to distinguish between three major global population groups. However, Chinese population was not included in Daniel’s research. In this study, DNA samples from three Chinese population groups living in west of China (Han ( n = 60), Uighur ( n = 60) and Tibetan ( n = 60)) were extracted and analyzed following Daniel’s procedure. The difference in biogeographic ancestry between the three Chinese population groups was compared. The consequence shows no significant difference between Chinese Han, Uighur and Tibetan populations. In summary, this 21-plex DIP panel is unable to distinguish the three Chinese populations. To characterize three groups in China, more AIM-DIPs are needed. [ABSTRACT FROM AUTHOR]