Semester

Spring

Date of Graduation

2010

Document Type

Thesis

Degree Type

MS

College

Eberly College of Arts and Sciences

Department

Biology

Committee Chair

Clifton Bishop

Abstract

With the advent of quantitative reverse-transcription real-time polymerase chain reaction, RNA has increasingly become an invaluable tool to forensic science investigations. For instance, previous studies have shown that the age of a biological stain could be estimated using differential decay rates of different RNA segments, providing a temporal link between the suspect and the crime. By co-isolating RNA and DNA from the same sample, both a genetic profile and various other analyses can be conducted. RNA samples are frequently treated with DNase prior to analysis to rid samples of contaminating DNA. Although all manufacturers of DNase claim no loss of RNA-derived signal, our results indicate that only those based upon heat inactivation of the DNase showed acceptable levels of signal loss. Loss of cDNA is not permissible for studies where levels of starting RNAs are crucial in obtaining accurate results. Ideally, primers and probes for estimating relative RNA levels will be designed to span exon-exon boundaries such that DNA contamination cannot provide a false signal. Unfortunately such primer and probe locations are not always possible. We also investigated the feasibility of using RNA degradation as a means of determining the approximate ex vivo age of semen samples. Our results indicate that, unlike bloodstains and other biological specimens, RNA contained within dried semen samples do not degrade in a predictable fashion after the first few days following deposition. After approximately 1--1.5 years, differential degradation was observed in dried semen stains therefore the use of this technique for exclusionary purposes may be possible. Our results also indicate that primers and probes designed to detect small segments of semen specific RNAs can be used to identify dried semen samples at least 1.5--2 years of ex vivo age. Liquid samples did show differential decay rates shortly after deposition. In conclusion, RNA will likely become a fundamental tool in the near future for forensic investigations but it must be kept in mind that the manufactures claims should be validated before all examinations.

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