mRNA在体液斑鉴定研究中内参基因的选择与应用

利用mRNA分析技术鉴定生物检材中各种体液斑迹的组织属性来源是近年来法医物证学的一个重要进展。终点逆转录PCR和实时荧光定量PCR技术是法医学实验室常用的mRNA检测技术,为保证实验结果的准确性,研究者通常选用组成性表达的管家基因作为内参,对不同样本提取RNA进行质控和标准化,以正确评估目标mRNA在各样本中的表达量。本文聚焦近年来使用mRNA分析技术进行体液斑迹组织属性来源鉴定的研究报道,对这些工作中内参基因的选择与应用效果进行综述。     

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.     

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification

Molecular‐based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT‐qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA‐specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.     

A novel multiplex assay system based on 10 methylation markers for forensic identification of body fluids

Identifying the source of body fluids found at a crime scene is an essential forensic step. Some methods based on DNA methylation played significant role in body fluids identification. Since DNA methylation is related to multiple factors, such as race, age, and diseases, it is necessary to know the methylation profile of a given population. In this study, we tested 19 body fluid-specific methylation markers in a Chinese Han population. A novel multiplex assay system based on the selected markers with smaller variation in methylation and stronger tissue-specific methylation were developed for the identification of body fluids. The multiplex assay were tested in 265 body fluid samples. A random forest model was established to predict the tissue source based on the methylation data of the 10 markers. The multiplex assay was evaluated by testing the sensitivity, the mixtures, and old samples. For the result, the novel multiplex assay based on 10 selected methylation markers presented good methylation profiles in all tested samples. The random forest model worked extremely well in predicting the source of body fluids, with an accuracy of 100% and 97.5% in training data and test data, respectively. The multiplex assay could accurately predict the tissue source from 0.5 ng genomic DNA, six-months-old samples and distinguish the minor component from a mixture of two components. Our results indicated that the methylation multiplex assay and the random forest model could provide a convenient tool for forensic practitioners in body fluid identification.     

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.     

Post-mortem interval estimation using miRNAs of road traffic accident cases: A forensic molecular approach

In forensic examination accurate estimation of post-mortem interval (PMI) is a challenging task, particularly in the advanced stages of decomposition. The existing methods (algor mortis, livor mortis, rigor mortis, putrefaction etc) used for estimating PMI rely on analyzing the physical, biochemical, and metabolic changes that occur in the corpse after death. While these methods have shown some level of effectiveness in estimating PMI during the early stages of decomposition, accurate estimation becomes increasingly challenging during the later stages of putrefaction when the body undergoes significant changes. Recently, microRNA (miRNA) profiling due to its relatively small size and stability has emerged as a promising tool in several areas of forensics. This study demonstrates the potential of miRNA for PMI estimation in advanced stages of death. In this study, miRNA-195, miRNA-206, and miRNA-378 were selected as target miRNAs and miRNA-1 as reference miRNA. Left ventricle tissue (5 g) of the heart from 20 forensic autopsies of traffic accident victims (18–32 years) were collected and processed. The samples were held at room temperature for eight different time intervals (12, 24, 48, 72, 96, 120, 168 and 196 h), and RNA was extracted from all the samples using Trizol-based RNA isolation protocol, followed by cDNA synthesis and amplification with commercially available specific miRNA probes in Real-Time PCR (RT-PCR), Ct was calculated. The result showed that miRNAs were associated with PMI. Over time, there were substantial changes in the Ct values of all three miRNAs, with significant reductions observed at 196 h compared to 12 h. miRNA-206 demonstrated significant changes at multiple time intervals, while miRNA-1 remained stable for up to 196 h and thus holds caas an endogenous marker. In conclusion, miRNA has the potential to serve as a valuable tool for estimating PMI, especially during the advanced stages of decomposition, when used in conjunction with established techniques. However, further validation of the study is required to obtain more accurate estimates of PMI.     

Evaluation of two DNA/RNA co-extraction methods for body fluid identification in forensics

Body fluid identification has become a field of interest in forensic casework as it can add value to particular investigative scenarios. Identifying the source of the biological material is not always an upfront task using conventional methods; therefore, profiling of specific mRNA markers can provide the answer. The implementation of RNA based analyses in forensic casework must focus on the quality and sensitivity of methods, starting with nucleic acid extraction, and without loss of DNA for STR profiling. In this work, two methods for DNA and RNA co-extraction were tested and compared: a commercial kit that uses a spin, mini column methodology, and a quick, simple nucleic acid isopropanol precipitation based protocol. Both methods simultaneously extract DNA and RNA, crucial for forensic casework and were tested in semen samples. Nucleic acid quantifications as well as purity assessment ratios (OD₂₆₀/OD₂₃₀ and OD₂₆₀/OD₂₈₀) were obtained by both methods to infer on the use of extracts in downstream applications such as PCR. The performance of the two tested protocols was further evaluated by analyzing two semen mRNA specific markers, PRM1 and SEMG1. When compared to the commercially developed kit, results suggest that the literature adapted protocol allowed more carryover of contaminants absorbing at 230 nm and 280 nm as the purity ratios were below the accepted standard ranges. Negative results for mRNA profiling supported the QC results obtained by spectrophotometry. On the hand, PRM1 and SEGM1 were positive in RNA samples extracted with the commercial kit.     

The development of a method for FISH identification of forensically relevant body fluids

Messenger RNA profiling is a useful confirmatory test for body fluid identification, but there are limitations to this method including sensitivity and the difficulty in linking body fluids with the corresponding DNA profile in mixed samples. We have developed a method for RNA suspension fluorescence in situ hybridisation (RNA S-FISH) for forensic-type samples using locked nucleic acid (LNA) probes. Vaginal and buccal epithelial cells have been the primary focus, with some preliminary work performed on leucocytes and seminal round cells. We have designed probes for the KRT10 messenger RNA and the micro RNA 891a. Using these probes, we have optimised a RNA S-FISH methodology and have successfully visualised these cell types using fluorescent microscopy.     

A molecular identification system for grasses: a novel technology for forensic botany

Our present inability to rapidly, accurately and cost-effectively identify trace botanical evidence remains the major impediment to the routine application of forensic botany. Grasses are amongst the most likely plant species encountered as forensic trace evidence and have the potential to provide links between crime scenes and individuals or other vital crime scene information. We are designing a molecular DNA-based identification system for grasses consisting of several PCR assays that, like a traditional morphological taxonomic key, provide criteria that progressively identify an unknown grass sample to a given taxonomic rank. In a prior study of DNA sequences across 20 phylogenetically representative grass species, we identified a series of potentially informative indels in the grass mitochondrial genome. In this study we designed and tested five PCR assays spanning these indels and assessed the feasibility of these assays to aid identification of unknown grass samples. We confirmed that for our control set of 20 samples, on which the design of the PCR assays was based, the five primer combinations produced the expected results. Using these PCR assays in a 'blind test', we were able to identify 25 unknown grass samples with some restrictions. Species belonging to genera represented in our control set were all correctly identified to genus with one exception. Similarly, genera belonging to tribes in the control set were correctly identified to the tribal level. Finally, for those samples for which neither the tribal or genus specific PCR assays were designed, we could confidently exclude these samples from belonging to certain tribes and genera. The results confirmed the utility of the PCR assays and the feasibility of developing a robust full-scale usable grass identification system for forensic purposes.     

The selection of endogenous genes in human postmortem tissues

Precisely determining the postmortem interval (PMI), which is crucial to criminal and forensic cases, is a research in which quantitative RT-PCR (also known as qRT-PCR or real-time RT-PCR) has been used to analyse gene expression levels and data normalisation should be required to eliminate the differences among the samples. Therefore, it is quite necessary to find stable molecular biological markers in PMI determination research. In this study, we compared nine commonly used endogenous markers (containing ACTB, GAPDH, B2M, U6, 18S rRNA, hsa-mir-1, hsa-mir-9, hsa-mir-194-1 and hsa-mir-203) in the 109 human tissue samples obtained from autopsy at the aim of finding stable markers in human tissues with consideration of the impact of parameters (PMI and cause of death). After RNA was extracted from four tissues (heart, brain, kidney, skin), the Ct values of nine endogenous markers were obtained by qRT-PCR and assessed by geNorm software. The results showed that U6, GAPDH and 18S rRNA were the suitable markers in our set of samples in various corpse conditions, that B2M and ACTB were reliable internal controls in heart tissue only, and that microRNAs had such high M values that they should not be chosen for endogenous control genes.     

Use of polymerase chain reaction of the HLA-DQ alpha system in forensic trace analysis]

The polymerase chain reaction (PCR) can be used for genetic typing of minute amounts of biological stains. This is achieved by in vitro amplification of a well-defined and genetically polymorphic human genomic DNA sequence. Using the HLA-DQ alpha system, a population study was carried out among 212 unrelated individuals of German origin. The usefulness of this system is discussed by presenting examples of its application in forensic casework, i.e. the analysis of mixed (male/female) body fluids as well as segregation studies on embryonic and paraffin-embedded tissue samples.     

DNA internal standard for the quantitative determination of hallucinogenic plants in plant mixtures

Here, we show a new, simple, and rapid SYBR Green-based Real-Time PCR assay for the quantification of hallucinogenic plants in plant mixtures. As a test plant, Salvia divinorum Epling & Játiva-M., a perennial herb belonging to the Lamiaceae family able to induce hallucinations, changes in perception, or other psychologically induced changes with similar potency as LSD, was used. The method was tested on seven mixtures 100/0%, 80/20%, 60/40%, 40/60%, 20/80%, 10/90%, 0/100% (w/w) S. divinorum versus a non-hallucinogenic plant, Salvia officinalis. Total DNA was extracted from samples and quantified by Real-Time PCR. Arabidopsis thaliana genomic DNA was added, as internal standard, at the beginning of each extraction. A new formula for the interpretation of Real-Time PCR data, based on the relative quantification of DNA extracted from mixture versus a reference DNA extracted from a known amount of pure S. divinorum, was developed. The results of this work show an almost perfect correspondence between Real-Time PCR-calculated weight and the weight estimated by an analytical weighted method, proving the effectiveness of this method for the quantitative analysis of a given species in a plant mixture.