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.     

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.     

microRNA Detection in Blood,Urine, Semen,and Saliva Stains After Compromising Treatments

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.     

基于miRNA相对表达量自动判别月经血和外周血

目的探索有效区分月经血和外周血的miRNA最优标记组合及最佳分类模型,并构建简便快速的自动化判别软件。方法对10种miRNA(miR-451a、miR-205-5p、miR-203a-3p、miR-214-3p、miR-144-3p、miR144-5p、miR-654-5p、miR-888-5p、miR-891a-5p、miR-124-3p)在200余份月经血和外周血样本中的相对表达量以实时荧光定量PCR检测,并以7种算法模型(核密度估计、K-最近邻、逻辑回归、线性判别分析、支持向量机、神经网络、随机森林)进行数据分析,选出鉴别效果最好的标记组合及算法模型,进而构建自动判别软件。结果月经血和外周血中差别最大的三种miRNA为miR-205-5p、miR-203a-3p和miR-214-3p,使用miR144-5p与上述miRNA中的一种或两种组合可达较好区分效果,其中基于miR-144-5p、miR-203a-3p和miR205-5p所形成的“最优特征项组合一”稳健性最强。7种算法模型中最佳分类模型为核密度估计模型,其次为逻辑回归模型。结论本研究建立的自动判别软件界面友好、使用简单,适合辅助法医检验关于月经血和/或外周血判别分析的计算,便利于法医物证工作,有较大的推广应用价值。     

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.     

microRNA的检测技术及其法医学应用前景

microRNA（miRNA）是一类由18～25个核苷酸构成的非编码小分子RNA，在转录后水平调控基因的表达。miRNA的表达具有高度保守性、时序性和组织特异性。随着其检测技术的逐渐成熟，miRNA被引入到法医学的研究中。新近研究表明，miRNA在法医学体液鉴定、种属鉴定和PMI推断等方面有一定的应用前景。本文简述miRNA检测技术的发展，并对其在法医学中的应用及前景进行综述，以期为相关研究及实践提供参考。     

A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification

We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice.     

Developmental Validation of RSID™-Saliva: A Lateral Flow Immunochromatographic Strip Test for the Forensic Detection of Saliva

Abstract:  Current methods for forensic identification of saliva generally assay for the enzymatic activity of α-amylase, an enzyme long associated with human saliva. Here, we describe the R apid S tain ID entification (RSID™-Saliva), a lateral flow immunochromatographic strip test that uses two antisalivary amylase monoclonal antibodies to detect the presence of salivary amylase, rather than the activity of the enzyme. We demonstrate that RSID™-Saliva is accurate, reproducible, and highly sensitive for human saliva; RSID™-Saliva detects less than 1 μL of saliva. The sensitivity of RSID^TM-Saliva allows investigators to sample a fraction of a questioned stain while retaining the majority for DNA-STR analysis. We demonstrate that RSID™-Saliva identifies saliva from a variety of materials (e.g., cans, bottles, envelopes, and cigarette-butts) and it does not cross-react with blood, semen, urine, or vaginal fluid. RSID™-Saliva is a useful forensic test for determining which evidentiary items contain saliva and thus may yield a DNA profile.     

磁珠直接吸附法对体态检材游离DNA的提取

目的采用磁珠直接吸附法对人体尿液、唾液、血液3种体态生物检材中的游离DNA进行提取检验,为法医物证中游离DNA的研究及检验工作提供参考。方法对3种生物检材采取离心吸取上清液的方法分离游离DNA,然后采用磁珠直接吸附法进行提取纯化,Identifiler-Plus试剂盒进行复合扩增后常规STR检测。结果在3种检材中均检出了游离DNA,其中血液中游离DNA检出率为100%,唾液为90%,尿液为70%。结论人体体态生物检材中存在游离DNA,同时磁珠直接吸附法可高效、快捷的提取生物检材中的游离DNA。     

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.     

人类岩藻糖基转移酶5特异性分布及其在精细胞的表达与定位

目的研究人类岩藻糖基转移酶5(fucosyltransferase 5,FUT5)的特异性分布及在精细胞的表达与定位。方法收集健康志愿者的精液(分离精细胞并提取精细胞膜蛋白)、阴道拭子、唾液及静脉血,应用免疫印迹方法检测FUT5在人类精细胞膜、精浆、阴道液、唾液及血清中的表达量,采用免疫荧光技术检测FUT5在精细胞中的表达与定位。结果免疫印迹方法结果显示FUT5在精细胞膜及血清中有较高表达,但在精浆、阴道液及唾液中未被检测到。免疫荧光实验结果显示FUT5主要存在于精细胞头部。表明人类精细胞膜存在一定表达量的特异性FUT5,可采用抗原-抗体反应分离精液和阴道液混合斑中的精细胞。结论人类FUT5表现出分布特异性,可应用到法医学性犯罪案件中混合斑的鉴定。     

Comparison of Catalytic and Immunological Amylase Tests for Identifying of Saliva from Degraded Samples

The stability of salivary α‐amylase is a critical factor in both catalytic and immunological method‐based forensic saliva identification. This study aimed to assess the sensitivity of catalytic and immunological tests on degraded saliva samples. Degraded saliva stains were prepared by microbial decomposition using humid soil. Salivary α‐amylase activity was catalytically detected both qualitatively and quantitatively using the Phadebas^® amylase test. As immunological methods, we conducted qualitative and quantitative tests using the RSID?‐saliva test and ELISA, respectively. Salivary α‐amylase activity of degraded samples (incubated at 37°C for 12 h) was significantly lower than that of controls in the quantitative tests. All the degraded samples obtained by the humid soil produced negative results in the Phadebas^® tests, but showed positive results in the RSID?‐saliva test and ELISA. These results suggest that immunological tests are effective for testing degraded saliva samples that have lost their enzymatic activity.     

创伤性脑损伤大鼠P300检测与海马microRNA表达变化

目的探讨创伤性脑损伤后大鼠海马microRNA的表达变化与认知功能障碍的相关性。方法 25只SD大鼠随机分为假手术组和伤后1h、1d、3d、5d组,采用Feeney自由落体法建立创伤性脑损伤动物模型。各组大鼠于设定的损伤时间点检测事件相关电位P300,应用基因芯片技术检测海马microRNA的表达情况,筛选特异性表达的microRNA。结果各组大鼠伤后P300的潜伏期延长,波幅下降,以伤后1d为甚。与假手术组相比,伤后各实验组microRNA表达谱具有显著差异。其中miR-21、miR-16、let-7b的表达与P300潜伏期变化存在显著相关,伤后1d为差异性表达的关键时间点。结论 microRNA可能在创伤性脑损伤后发生的认知功能障碍中发挥调控作用,有望为脑损伤后认知功能的法医学鉴定提供新的思路。     

唾液中滥用药物分析及其与血液中药物浓度的相关性

唾液是一种成分简单、易于采集的体液,某些药物在唾液中的浓度可以反映其血药浓度。本文分析了滥用药物进入唾液的机制和影响因素,综述了唾液中滥用药物分析时样品的采集、前处理和检测方法以及唾液与血液中药物浓度的相关性。认为唾液是临床和法医学方面很有价值的分析样品,用唾液中滥用药物浓度来推测血药浓度具有一定的法医学意义。     

Comparison of modern techniques for saliva screening

Saliva stains present a unique challenge in the forensic setting, often challenging the analyst to weigh the value of presumptive indication of the fluid versus the potential for DNA analysis to yield identification information. There are many situations in which determining the presence of a body fluid is probative and further corroborates DNA evidence. That said, even a minute portion of sample consumed by a screening test could mean the difference between a full, partial, or null profile obtained through DNA analysis. The basis of presumptive testing or screening of saliva has historically been based on the presence of amylase, a component found in relatively high concentrations in human saliva versus other body fluids and substances. Though the current available methods for the screening of saliva in a forensic application have grown in number, the popularity of these methods seemingly has not. This study attempts to identify a specific and sensitive saliva screening test by comparing three modern techniques--the recently released SALIgAE, Phadebas, and starch-iodine mini-centrifuge test--on the basis of sensitivity, specificity, mixtures, and simulated casework samples while also considering sample consumption. The Phadebas method for presumptive saliva testing detected dilutions of neat saliva down to 1:200 versus considerably less sensitive results with SALIgAE and the starch-iodine mini-centrifuge test. Utilizing a screening test with a high degree of sensitivity, such as Phadebas, allows an analyst to gain a maximum amount of information in the form of body fluid indication and DNA results because of the consumption of a small portion of sample.     

Screening and identification of tissue-specific methylation for body fluid identification

Identification of body fluid stains can bring important information to crime case. Recent research in epigenome indicates that tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to the type of cell or tissue, which makes it possible to identify body fluid based on analysis of DNA. This study screened and identified tDMRs from genome for forensic purpose. DNA samples from blood, saliva, semen, and vaginal fluid were analyzed by methylation sensitive represent difference analysis and Sequenom Massarray^® quantitative analysis of methylation. Six blood-specific tDMRs were obtained. Two tDMRs display blood-specific hypomethylation, and four tDMRs show blood-specific hypermethylation. These tDMRs may discriminate blood stain from other body fluids. The result indicated that tDMRs could become potential DNA markers for body fluid identification.     

法医物证学miRNA分析的研究进展

在种属和体液鉴定及降解检材等特殊案件的分析时,转录水平的miRNA所具有的生物属性及表达特点,使其能够发挥基因组DNA所不具备的价值。本文通过概述法医物证学miRNA研究的现状,对法医miRNA分析的研究策略和法医物证学应用前景进行了综述,以期为法医miRNA分析的应用研究提供借鉴。