Keywords

mRNA profiling, body fluid identification, tissue identification, multiplex RT-PCR, multiplex qPCR, RNA stability/degradation

Abstract

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.

Notes

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Graduation Date

2005

Semester

Fall

Advisor

Ballantyne, Jack

Degree

Doctor of Philosophy (Ph.D.)

College

Burnett College of Biomedical Sciences

Department

Chemistry

Degree Program

Biomolecular Sciences

Format

application/pdf

Identifier

CFE0000862

URL

http://purl.fcla.edu/fcla/etd/CFE0000862

Language

English

Release Date

January 2007

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

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