In theory, RNA expression patterns, including the presence and relative abundance of particular RNA species, provide cell and tissue specific information that could be of use to forensic scientists. An mRNA based approach could allow the facile identification of the tissue components present in a body fluid stain and conceivably could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory. Some of the potential advantages include greater test specificity, and the ability to perform simultaneous analysis using a common assay format for the presence of all body fluids of forensic interest. In this report, the recovery and stability of RNA in forensic samples was evaluated by conducting an in-depth study on the persistence of RNA in biological stains. Stains were prepared from blood, saliva, semen, and vaginal secretions, and were exposed to a range of environmental conditions so that the affects of different light sources, temperatures, and environments could be assessed. Using the results from quantitation and sensitivity studies performed with pristine forensic stains, the RNA stability of samples which were collected over a period of 1 day to 1 year for blood, saliva, and vaginal secretion stains and for up to 6 months for semen stains were analyzed. The extent of RNA degradation within each type of body fluid stain was determined using quantitation of total RNA and reverse transcriptase polymerase chain reaction (RT-PCR) with selected housekeeping and tissue-specific genes. The results show that RNA can be recovered from biological stains in sufficient quantity and quality for mRNA analysis. The results also show that mRNA is detectable in samples stored at room temperature for at least one year, but that heat and humidity appear to be very detrimental to the stability of RNA.

Vaginal fluid is most often found at crime scenes where a sexual assault has taken place or on clothing or other items collected from sexual assault victims or perpetrators. Because the victim is generally known in these cases, detection of vaginal fluid is not a matter of individual identification, as it might be for semen identification. Instead, linkages can be made between victim and suspect if the sexual assault was carried out digitally or with a foreign object (e.g., bottle, pool cue, cigarette, handle of a hammer or other tool, etc.). If such an object is only analyzed for DNA and the victim is identified, the suspect may claim that the victim’s DNA is present because she handled and/or is the owner of the object and not because it was used to sexually assault her; identification of vaginal fluid residue would alleviate such uncertainty. Most of the research conducted thus far regarding methods for the identification of vaginal fluid involves mRNA biomarkers and identification of various bacterial strains.1-3 However, these approaches require extensive sample preparation and laboratory analysis and have not fully explored the genomic differences among all body fluid RNAs. No existing methods of vaginal fluid identification incorporate both high specificity and rapid analysis.4 Therefore, a new rapid detection method is required. Surface-enhanced Raman spectroscopy (SERS) is an emerging technique with high sensitivity for the forensic analysis of various body fluids. This technique has the potential to improve current vaginal fluid identification techniques due to its ease-of-use, rapid analysis time, portability, and non-destructive nature. For this experiment, all vaginal fluid samples were collected from anonymous donors by saturation of a cotton swab via vaginal insertion. Samples were analyzed on gold nanoparticle chips.4 This nanostructured metal substrate is essential for the large signal-enhancement effect of SERS and also quenches any background fluorescence that sometimes interferes with normal Raman spectroscopy measurements.5 Vaginal fluid SERS signal variation of a single sample over a six-month period was evaluated under both ambient and frozen storage conditions. Vaginal fluid samples were also taken from 10 individuals over the course of a single menstrual cycle. Four samples collected at one-week intervals were obtained from each individual and analyzed using SERS. The SERS vaginal fluid signals showed very little variation as a function of time and storage conditions, indicating that the spectral pattern of vaginal fluid is not likely to change over time. The samples analyzed over the span of one menstrual cycle showed slight intra-donor differences, however, the overall spectral patterns remained consistent and reproducible. When cycle spectra were compared between individuals, very little donor-to-donor variation was observed indicating the potential for a universal vaginal fluid signature spectrum. A cross-validated, partial least squares – discriminant analysis (PLS-DA) model was built to classify all body fluids, where vaginal fluid was identified with 95.0% sensitivity and 96.6% specificity, which indicates that the spectral pattern of vaginal fluid was successfully distinguished from semen and blood. Thus, SERS has a high potential for application in the field of forensic science for vaginal fluid analysis.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22378号 / 医科博第108号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 羽賀 博典, 教授 松村 由美 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Blood is considered one of the most important types of forensic evidence found at a crime scene. The use of surface-enhanced Raman spectroscopy (SERS) provides a potentially non-destructive and highly sensitive technique for the confirmation of blood and this method can be applied using a portable Raman device with quick sample preparation and processing. Crime scenes are inherently complex and the impact of SERS analysis provides easy use and practical application for in-field sample analysis. SERS is one of the few confirmatory techniques employed for the identification of blood at a crime scene or in the forensic laboratory. This method is able to distinguish between blood and other body fluids by collecting a SERS spectrum from a sample placed on a surface that has been embedded with gold nanoparticles (AuNPs). The AuNPs create an electric field surface enhancement that produces an intense molecular vibrational signal, leading to a SERS enhancement. The SERS enhancement allowed for sensitive blood detection at dilutions greater than 1:10,000. A stain transfer method to the SERS substrate was optimized by extracting dried bloodstains with water, saline, and various acid solutions. Fifty percent aqueous acetic acid solutions was found to be the most efficient in retaining the blood components and releasing the hemoglobin component of blood for detection. The SERS spectrum of blood is a robust signature of hemoglobin that does not significantly change between donors nor over time. Characteristic peaks for the identification of blood are 754, 1513, and 1543 wavenumbers (cm-1), attributed to a pyrrole ring breathing mode (15) and two Cβ-Cβ stretches (11, 38), respectively. These key SERS peaks, high sensitivity, and signal enhancement are favorable when compared to normal Raman spectroscopy. A quick and easy-to-use procedure for on-site sample analysis for the detection of blood on different substrates was developed and applied on a portable Raman device. Various nonporous and porous substrates including glass, ceramic tile, cotton, denim, fleece, nylon, acetate, wool, polyester, wood, and coated wood yielded strong results for identification of bloodstains. In addition, different commercial and in-house SERS substrates were tested to determine effectiveness for the detection and identification of blood. SERS identification of blood for forensic work is a potentially non-destructive and portable tool that can be applied for quick and easy examination of evidence at a crime scene. The high sensitivity and selectivity of SERS provides a robust spectroscopic signature that aids in the confirmation of blood, even when it is not visible to the naked eye. It is a more favorable method when compared to current presumptive and confirmatory tests for blood and can be applied to stains on different SERS substrates and a variety sample surfaces for universal testing.

Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs can be determined in a stain or tissue sample recovered at the crime scene, it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automated analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis using reverse transcriptasepolymerase chain reaction assay (RT-PCR). We have identified sets of candidate tissuespecific genes for body fluids and tissues of forensic interest, namely blood, saliva, semen, vaginal secretions, menstrual blood, urine, skin, muscle, adipose, and brain. We also report the identification of a new housekeeping gene for use in mRNA based assays. Select body fluid-specific genes have been incorporated into multiplex PCR and real-time PCR assays. These assays allow for the positive identification of blood, saliva, semen,vaginal secretions, and/or menstrual blood in a stain. The final task of this work was the molecular characterization of mRNA degradation patterns in biological stains, which not only has fundamental importance in possibly revealing mRNA degradation pathways in dried biological stains, but may ultimately lead to better assay design strategies for mRNA markers for forensic use. An mRNA-based approach described in this report could allow the facile identification of the tissue components present in a body fluid stain and could conceivably supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.

Prostate cancer is the second-most diagnosed and fatal carcinoma for males in the United States, and better diagnostic markers and potential therapies are needed. microRNAs are small, single-stranded RNA molecules that affect protein expression at the translational level, and dysregulation can dramatically affect cell metabolism. Comparison of 736 microRNA expression levels between the poorly metastatic SV40T immortalized prostate epithelial cell line P69 to its highly tumorigenic and metastatic subline M12 identified 231 miRs that were overexpressed and 150 miRs that showed loss of expression in the M12 cell line. Further evaluation of fourteen identified miRs was accomplished using other prostate cell lines as well as laser-capture microdissected prostate samples. Inhibition of miR-147b was found to affect proliferative, migratory and invasive capabilities of M12 cells, and reduced tumour growth in nude athymic mice. AATF, an activator of the cell-cycle inhibitor p21, was identified as a target. Overexpression of miR-9 was found to affect the epithelial to mesenchymal transition through suppression of e-cadherin, a protein characterized as lost in EMT, as well as suppression of SOCS5, an attenuator of JAK-STAT signaling. Inhibition of miR-9 resulted in reduction of migratory and invasive potential, and significant reduction of tumorigenesis and metastases in male nude athymic mice. miR-17-3p was previously identified as down-regulated in prostate cancer and loss of miR-17-3p shown to cause vimentin transcriptional activation. Reverse phase microarray analysis (RPMA) identified c-KIT as a potential second mRNA target for miR-17-3p. miR-17-3p was shown to modulate not only protein levels, but also messenger RNA levels of c-KIT. Four miR-17-3p binding sites in the c-KIT mRNA were identified. Thus, a number of microRNAs involved in prostate cancer were identified, and their targets found to be highly relevant to tumour progression and could potentially be used as targets for therapy or diagnostics. Stability of microRNAs in forensically relevant biological fluids was evaluated through heat treatment, ultraviolet radiation, and chemical treatment. The dried body fluids showed some susceptibility to harsh treatment, but in most cases microRNAs were still detectable in the samples. microRNAs could represent a highly stable species for body fluid identification methods in forensic science.