circRNA应用于体液斑鉴定的技术可行性研究

目的利用circ RNA检测技术,探索circ RNA分子应用于体液斑迹鉴定的可行性。方法制备精液、唾液、阴道分泌物三种体液斑迹样本,以Qiagen RNeasy Micro试剂盒提取总RNA,经RNase R消化后得到circ RNA,进行逆转录PCR扩增,琼脂糖凝胶电泳检测分析。结果制备的体液斑迹样本均可检测到circ RNA分子,提示circ RNA普遍存在于法医常见体液斑迹中,具备一定的应用价值。结论 circ RNA的检测可与现有DNA检测技术兼容,利用其组织特异性,可成为体液斑迹鉴定的新标记,具备重要的研究价值。     

mRNA在体液斑迹鉴定与组织来源推断中的应用

体液斑迹鉴定及其组织来源推断一直是法医学研究的重要方向之一,其传统的检验方法存在诸如假阳性率高、检材易破坏等缺陷,亟需更高效的确证实验。高度分化的体细胞表达特异的m RNA分子,可推断其来源:外周血、月经血、精液、唾液、阴道分泌物、接触斑等,且灵敏度、特异度及保存时间相对较为理想,是未来用于体液斑迹鉴别的理想遗传标记。本文对m RNA在体液斑迹鉴定和组织来源推断方面的应用及其前景进行综述。     

血痕特异性mRNA标记研究

血痕是案发现场尤其是命案中比较常见的生物物证,而血痕的正确组织来源推断是当前鉴定工作中急需解决的问题。随着法医物证学的不断发展,以mRNA(messenger RNA)为基础的体液斑迹组织来源鉴定技术作为一种不同于传统血痕免疫学检测的新型方法,已经越加显示其独特的优越性。在该技术的基础上,实现共同提取生物物证的RNA与DNA的目标,建立体液斑迹鉴定与DNA分型兼容的方法,有利于现场重建,提高生物物证的证据效力,完善证据链。本研究建立了一个包括血痕总RNA提取、逆转录、荧光特异引物扩增、遗传分析仪电泳检测分析等步骤的血痕来源推断技术平台。实验共采集制备了40份的中国人群(女性)外周血、16份月经血样本,筛选了5个外周血标记:HBA、HBB、GYPA、SPTB、ALAS2,2个月经血标记:MMP7、MMP11,构建了一个囊括外周血、月经血特异标记的荧光复合扩增体系。结果显示mRNA技术为基础的鉴定血痕来源的方法是可行的,并且建立了血痕RNA检验的遗传分析仪结果判读方法。     

大鼠皮肤挫伤后NF-KB mRNA表达与损伤时间关系

目的 应用实时定量PCR技术检测大鼠皮肤挫伤后NF-～B mRNA时序性表达规律,分析其与损伤时间的关系.方法 制备大鼠皮肤挫伤模型,于伤后0.5,1,6,12,18,24,30h取材,一步法提取总RNA,检测其完整性和浓度,按照0.4μg总RNA量逆转录合成第一链cDNA,以rip32为内参基因进行荧光定量检测,采用JACt法比较其与正常皮肤组织NF-kB mRNA表达的倍数关系.结果 皮肤挫伤组织中NF-KB mRNA伤后0.5h表达显著增强(P<0.05),6h出现高峰,12h降至正常水平,随后略有回升.结论 大鼠皮肤挫伤组织NF-KB mRNA的表达,随着损伤经过时间的延长呈规律性变化.     

体液斑迹遗留时间推断研究现状与展望

现场体液斑迹的遗留时间是案件侦破的重要线索,对其进行准确的推断,可以确定案发时间,有效缩小警方的侦查范围。近年来,随着仪器分析技术、高通量测序技术的不断进步,推断现场法医物证遗留时间的研究取得一定进展。本文聚焦现场最常见的物证类型之一体液斑迹,从血红蛋白及其衍生物、RNA、微生物、酶以及昼夜节律性标记物等方面梳理了体液斑迹遗留时间的研究成果,并从似然比的角度,就如何实现遗留时间的精准推断进行了展望。     

DNA甲基化在组织/体液来源鉴定中的研究进展

对可疑生物样本的组织/体液进行来源鉴别是重建犯罪现场、推断犯罪性质等侦查活动中极为重要的一环。对表观遗传学理论的研究证明运用基因组中存在的组织特异性DNA甲基化差异位点(t DMRs)可以对组织/体液进行来源鉴别。本文旨在通过对近年来DNA甲基化在法医学领域用于鉴定人体组织/体液来源方面的研究成果进行阐述,试图用所得到的信息来分析DNA甲基化作为一种组织/体液鉴定遗传学标记的可能性、优劣点及其应用价值和发展前景,以期能为法医工作者的相关研究及实践提供参考。     

利用mRNA检验确证无精子症精斑1例

正近年来,利用mRNA进行生物物证体液斑迹的研究已经取得一定的进展,其在DNA鉴定工作中发挥的作用也越来越被重视。本实验室受理了1起强奸案,通过mRNA的检测分析,为刻画嫌疑人提供了依据,现报道如下。1案例资料1.1简要案情2015年4月某强奸案中,现场提取到有疑似精斑的床单1件,斑迹经人精P30试纸条检测为阳性。用差异裂解法提取检材DNA,同时检测上清液和沉     

RNA指标在死后人体皮肤组织中稳定性研究

目的探讨多种RNA指标的表达水平在死后人体皮肤组织中的稳定性。方法收集死亡时间(PMI)明确的人体皮肤组织样本10例,提取总RNA,运用实时荧光定量PCR(qRT-PCR)技术检测ACTB、GAPDH、18S、miR-203、5S、1et-7a、RPS29、U6共计8个RNA指标的表达水平。geNorm软件评估各RNA指标表达水平的稳定性,挑选出稳定的内参指标并分析其表达水平与相关因素(年龄、性别、死因)的关系。结果人体皮肤组织中5S、U6和miR-203表达水平最为稳定,其平均表达量与年龄、性别、及死因无关(P0.05)。结论 5S、U6和miR-203可以作为qRT-PCR方法研究PMI的理想内参指标。     

大鼠皮肤挫伤后NF-κB mRNA表达与损伤时间关系

目的应用实时定量PCR技术检测大鼠皮肤挫伤后NF-κB mRNA时序性表达规律，分析其与损伤时间的关系。方法制备大鼠皮肤挫伤模型，于伤后0．5，1，6，12，18，24，30h取材，一步法提取总RNA，检测其完整性和浓度，按照0．4μg总RNA量逆转录合成第一链cDNA，以rlp32为内参基因进行荧光定量检测，采用△Ct法比较其与正常皮肤组织NF-κB mRNA表达的倍数关系。结果皮肤挫伤组织中NF-κB mRNA伤后0．5h表达显著增强（P〈0．05），6h出现高峰，12h降至正常水平，随后略有回升。结论大鼠皮肤挫伤组织NF-κB mRNA的表达，随着损伤经过时间的延长呈规律性变化。     

大鼠骨骼肌早期死亡时间推断的内参基因筛选

目的死亡时间推断是法医病理学实践的重点。运用mRNA定量技术进行死亡时间推断已成为目前研究的热点。在进行mRNA指标挑选时,为了排除其他干扰因素的影响,实验数据必须要经过内参指标的标准化。因此,内参基因的选择对实验结果具有重要影响。方法 SD大鼠颈椎脱臼处死后置于4℃、15℃、25℃、35℃环境温度下,在死后0h、6h、12h、18h、24h五个时间点取骨骼肌组织。利用实时荧光定量PCR技术测定常用内参基因Ct值,并使用三种内参评估软件(geNorm、NormFinder、BestKeeper)综合分析内参稳定性。结论综合三种内参评估软件结果,在低温条件下,RPS29、RPL13是较合适的内参,而在高温条件下,5SrRNA、18SrRNA是较合适的内参。在35℃时,GAPDH、HPRT、PGK1、UBC、B2M与死亡时间有明显相关性。     

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”.     

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.     

Messenger RNA profiling: a novel method for body fluid identification by real-time PCR

Conventional methods for the identification of different body fluids like blood, semen and saliva from biological stains involve immunological or enzymatic detection of certain proteins. In this study, we investigated potential RNA markers with the aim of developing Real-Time polymerase chain reaction (PCR) based methods to allow differentiation between several body fluids. Total RNA samples from artificially stained swabs and from various pieces of evidence from case work were extracted, amplified and analyzed with several RNA markers. Three assays detecting the body fluids of interest were selected: hemoglobin-alpha locus 1 (HBA), kallikrein 3 (KLK) and mucin 4 (MUC). With this approach, we demonstrate that specific Real-Time PCR assays are useful in identifying the source of the biological stain. Furthermore, RNA profiling of various body fluids was even possible on samples stored over a long period of time at ambient temperature. The stability and sensitivity of the applied method outlines a novel application for Real-Time PCR within the forensic field.     

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.     

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.     

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.     

Validation study of endogenous reference genes for normalization of quantitative real time PCR data in post mortem skin tissue

Gene expression profiling may provide insights into the molecular mechanisms underlying wound skin repair contributing to applications in the field of post mortem forensic wound age estimation. Quantitative real-time PCR (qPCR) is the most sensitive technique for gene expression studies but represents a complex procedure with several requirements for accurate data analysis. One of the major pre-requisites is the selection and validation of appropriate endogenous control genes for normalization in a given set of samples. In this study, the gene expression stabilities of ten endogenous controls ACTß, ß2M, PPIA, GAPDH, HPRT1, PGK1, SDHA, TBP, UBC, and YWHAZ were evaluated in two sets of samples: post mortem human wounded and unwounded skin tissue aimed for subsequent wound age estimation studies. The most stable genes were determined by calculating a gene expression normalization factor for each sample based on the geometric mean of the ten selected genes using the reference gene validation software, geNorm. In uninjured skin, the most stable endogenous controls were YWAZ, with the highest stability, followed by PGK1 and GAPDH. In the wounded samples, GAPDH was the most stable gene followed by PGK1, ACTß and HPRT1. Both groups revealed some differences relative to reference gene expression stabilities, nevertheless, PGK1 and GAPDH could serve as two possible common reference genes for qPCR data normalization as both were identified as stable genes in wounded and unwounded skin tissues. The differences of expression stabilities of the reference genes identified in both groups support the need for accurate normalization of gene-expression levels previous to qPCR studies.