Application of mRNA Expression Analysis to Human Blood Identification in Degenerated Samples that were False‐negative by Immunochromatography,,

Forensic laboratories are often faced with cases in which methamphetamine hydrochloride‐mixed blood is unable to be identified as human blood by immunochromatography against human hemoglobin A0. The application of mRNA expression analysis to samples that showed a false‐negative with immunochromatography was investigated as an alternative approach that did not depend on the antigen–antibody reaction. Real‐time PCR was used to examine the expression levels of blood markers such as glycophorin A, spectrin beta, and hemoglobin beta. Hemoglobin beta was the only marker that was specifically detected in blood, while glycophorin A was useful for determining human specificity. Hemoglobin beta showed good detection sensitivity and was detectable in 37‐year‐old blood stains. Hemoglobin beta was exclusively detectable in methamphetamine hydrochloride‐mixed blood stains. Detergents and disinfectants did not significantly influence mRNA markers. The proposed mRNA expression analysis was suitable for human blood identification as an alternative method to immunochromatography.     

Cutaneous (tPA) and Skeletal (TnI) mRNA as Markers of Aging in Contused Wound

Wound age estimation is one of the most important forensic aspects. Troponin I (TnI) and many cytokines, for example, tissue plasminogen activator (tPA), are involved in wound inflammation and healing. Skeletal (TnI) and cutaneous (tPA) mRNA was detected using real‐time PCR in 25 female albino rats. They were divided into 5 groups: control and 4 injured groups. Injured groups were sacrificed 1, 6, 24, and 30 h after inflicting contused wound. The expression levels of cutaneous (tPA) were decreased significantly at 1, 6, and 30 h after contusion (71.7%, 30.7 and 16.9%), while the expression levels of skeletal (TnI) were increased significantly at 1 and 6 h post‐traumatic, then they gradually decreased until reaching normal levels at 24 h and assumed significantly lower levels at 30 h postcontusion. These results suggested that the determination of cutaneous (tPA) and skeletal (TnI) mRNA levels was useful for wound age estimation.     

Bloodstain pattern analysis & Bayes: A case report

The findings from a bloodstain pattern analysis (BPA) may assist in formulating or falsifying scenarios that are considered in the investigative stages of a criminal investigation. When a case proceeds to trial the bloodstain pattern expert may be asked about the relevance of their findings given scenarios that are proposed by the prosecution and defense counsel. Such opinions provided by an expert are highly relevant to police investigation or legal proceedings, but the reasoning behind the opinion or implicit assumptions made by the expert may not be transparent.A proper framework for the evaluation of forensic findings has been developed since the late twentieth century, based on the hierarchy of propositions, Bayesian reasoning and a model for case assessment and interpretation. This framework, when implemented in casework, mitigates some of the risks of cognitive biases, and makes the reasoning and scientific basis for the opinion transparent. This framework is broadly used across forensic science disciplines. In this paper we describe its application to the field of BPA using a case example from the Netherlands Forensic Institute (NFI).     

鸭病毒性肝炎病毒RNA聚合酶在真核细胞中的表达及亚细胞定位

为研究鸭病毒性肝炎病毒RNA依赖性RNA聚合酶的分子生物学特性及其在细胞中的定位,构建了RNA依赖性RNA聚合酶与增强型绿色荧光蛋白融合表达的真核重组表达质粒;然后用脂质体介导转染MDCK细胞。分别使用荧光显微镜、激光共聚焦显微镜以及4,6-二脒基-2-苯基吲哚(DAPI)染色和蛋白免疫电泳试验等方法检测了RNA依赖性RNA聚合酶在细胞中的表达和亚细胞定位情况。结果表明,RNA依赖性RNA聚合酶能与增强型绿色荧光蛋白一起在MDCK细胞中良好表达,且主要分布于细胞核中,Western-blot分析结果显示,融合蛋白在80ku处出现阳性条带,说明表达的外源蛋白具有免疫活性。表明成功构建了RNA依赖性RNA聚合酶与增强型绿色荧光蛋白融合表达载体,并在真核细胞内实现了表达,通过检测荧光蛋白,发现RNA依赖性RNA聚合酶主要在细胞核中发挥复制酶的功能。     

Routine forensic use of the mitochondrial 12S ribosomal RNA gene for species identification

Since July 2004, Mitotyping Technologies has been amplifying and sequencing a approximately 150 base pair fragment of mitochondrial DNA (mtDNA) that codes for 12S ribosomal RNA, to identify the species origin of nonhuman casework samples. The approximately 100 base pair sequence product is searched at http://www.ncbi.nlm.nih.gov/BLAST and the species match is reported. The use of this assay has halved the number of samples for which no mtDNA results are obtained and is especially useful on hairs and degraded samples. The availability of species determination may aid forensic investigators in opening or closing off lines of inquiry where a highly probative but challenging sample has been collected.     

Evaluation of Tamm‐Horsfall Protein and Uroplakin III for Forensic Identification of Urine

Abstract: In this study, Tamm‐Horsfall protein (THP), a major component of urinary protein, and uroplakin III (UPIII), a transmembrane protein widely regarded as a urothelium‐specific marker, were evaluated for forensic identification of urine by ELISA and/or immunohistochemistry. THP was detected in urine, but not in plasma, saliva, semen, vaginal fluid, or sweat by the simple ELISA method developed in this study. In addition, most aged urine stains showed positive results. The urine specificity of THP was confirmed by gene expression analysis. Therefore, as reported previously, ELISA detection of THP can be used as a presumptive test for urine identification. UPIII was specific for immunohistochemical staining of cells in centrifuged precipitate of urine. However, ELISA and RT‐PCR for UPIII were not specific for urine. UPIII may be applicable for forensic urine identification by immunohistochemistry.     

死亡大鼠组织中总RNA提取的质量控制及方法评价

目的探讨不同商品化试剂盒在提取组织总RNA过程中的优劣与适用范围,为实验室的质量控制提供依据。方法采用以膜提取技术为基础（SV total RNA isolation system）和以异硫氰酸胍/苯酚法为原理（TRIzol总RNA提取试剂盒）的两种试剂盒对死亡大鼠心、肝、脾、肺、肾、脑组织进行总RNA的提取与评价鉴定,并使用琼脂糖变性凝胶电泳和2100芯片生物分析仪对提取的总RNA进行完整性鉴定和质量控制评价。结果两种商品化试剂盒提取的总RNA纯度与得率均可满足实验要求。各组织脏器中总RNA得率依次为脾脏＆gt;肝脏＆gt;肾脏＆gt;大脑＆gt;肺脏＆gt;心脏。结论与TRIzol总RNA提取试剂盒相比,SV Total RNA Isolation System提取时对操作人员熟练度要求较低,2100芯片生物分析仪有望替代凝胶电泳技术用于总RNA提取时的质量控制。     

反义RNA重组逆转录病毒逆转录套式PCR检测方法的建立

为探求一种快速检测反义RNA重组逆转录病毒效价的新方法,用脂质体介导法将反义RNA重组逆转录病毒载体pLXSA、pLXSB分别转染包装细胞系PA317,经G418筛选,获得数个稳定的产毒细胞克隆,择优挑选7个细胞克隆扩增培养,收集细胞上清,用异硫氰酸胍-酚一步法提取病毒RNA,进行逆转录套式PCR,检测细胞上清中的反义RNA重组逆转录病毒效价,同时用传统方法(小鼠成纤维放大法)进行测定。2种方法检测结果的比较表明,逆转录套式PCR法的特异性和敏感性均高于小鼠成纤维放大法,是一种敏感性高、特异性强、操作简便、快速的检测逆转录病毒效价的新方法。     

应用RNA干扰技术对猪蛔虫性别相关基因功能的初步研究

针对编码猪蛔虫雌虫卵巢蛋白基因的EST序列设计了1对特异引物及连接T7启动子的引物,经PCR扩增,获得5′端连有T7启动子的双链DNA,在RNA聚合酶的作用下,转录合成dsRNA。通过浸泡的方式将dsRNA导入秀丽新杆线虫中,在浸泡后的5个不同的时间点,采用RT-PCR检测虫体浸泡后靶基因被干扰的效果。试验结果表明,在浸泡后的8~29 h能检测到同源靶标的存在;而在浸泡后的43~57 h不能检测到靶标RNA。另外,试验组没有观测到虫体明显的表型变化,而对照组在浸泡28 h后,发现部分线虫体内有发育成形的虫卵。初步认为导入虫体的dsRNA发生了干扰效应。     

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.