Semester

Fall

Date of Graduation

2010

Document Type

Thesis

Degree Type

MS

College

Eberly College of Arts and Sciences

Department

Biology

Committee Chair

Clifton Bishop

Abstract

In forensic science, polymerase chain reaction (PCR) became an indispensable tool given the limited amount of biological samples usually encountered at crime scenes. DNA analysis is used to identify the source of biological samples typically obtained from a single hair, or droplet of blood. Determining the source of the biological evidence can provide a spatial link, thereby including or excluding a suspect at a crime scene or other locations related to a crime investigation. In spite of the great efficiency in human identification, DNA analysis cannot provide any information regarding time of deposition of the sample. The ability to establish a temporal connection reveals key information for crime scene reconstruction and evidence interpretation; this is especially true when determining if the DNA sample found at the crime scene was left at the moment of the crime or originated from an unrelated event. Estimating the age of the biological sample would be particularly important in cases where the victim and suspect are known to have a personal relationship. The development of quantitative reverse-transcription real-time polymerase chain reaction, has stimulated scientist to explore the potential use of RNA as a forensic tool. Multiple studies have reported the use of RNA analysis on body fluid identification, age determination of injuries and wounds and for post-mortem interval (PMI). Previously, our laboratory has shown that the estimation of age of a biological sample can be determined by measuring the degradation rates of two different RNA segments using real-time RT PCR method. In addition, it has also been demonstrated that, under controlled conditions, RNA decay proceeds in a predictable fashion. However, it is unrealistic to expect that in real crime scenes the biological sample will be exposed to an invariable environment. We investigated the environmental effects on beta-actin and 18S RNAs decay, more specifically; the effects of fluctuating temperatures and humidity by exposing bloodstain samples in two different rooms at WVU's Crime House One during a 90 day period. Daily temperature and relative humidity were recorded in each room. We also investigated the potential use of outdoor temperature to predict indoor temperature. In addition, we investigated the incorporation of accumulated degree days (ADD) into RNA degradation analysis in order to take into account the temperature changes in a non-controlled environment. We believe this will allow for a more accurate and reliable method for estimating time of deposition of blood samples. Our results indicate that the environmental conditions had an effect on the degradation rate of both beta-actin and 18S RNAs. The basement environment presented high but generally constant temperature and RNA decay occurred in a linear, predictable fashion. However, the accuracy of our estimation method was extremely decreased in a highly variable environment (attic). This suggests that our assay would only be accurate if there is no extreme fluctuation in temperature. Finally, our results show the importance of knowing the environmental conditions for an accurate estimation of time of deposition and how the data interpretation could be affected, if this information is unknown. After the 90 day exposure period, the basement had an ADD value of 1,496.047 while the attic had an ADD of 508.967 and the airport ADD was 143.111. Thus, using the ADD from one of these environments to estimate time of deposition on the other could lead to estimating the age of the sample as "older" or "younger" then it's true value.

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