Transcriptome Sequencing of Forensically Relevant Biological Fluids and Tissues to Obtimize Degradation Analysis for Sample Age Estimation

As submitted by the proposer: Research on the forensic applications of RNA analysis has increased greatly in the last decade. Defined uses of RNA in forensic analysis include the use of RNA to identify tissue type, determine sample age, and play a role in molecular autopsies. Although recent research has indicated many possible forensic applications of RNA analysis, many questions remain concerning the behavior of RNA in degraded and limitedly available samples. Specifically, there remains to be a thorough understanding of the differing patterns and rates of RNA degradation in post-mortem and deposited samples. Thus, choosing suitable RNA markers for evaluating the approximate age of a forensic sample can be problematic. Development of a reliable and accurate molecular assay for the determination of sample age (time-since deposition of a biological sample and/or post-mortem interval) will play a critical role in helping investigators establish the timeline of events that surround a crime. The purpose of this research is to evaluate RNA degradation in forensically relevant biological sample types (blood, saliva, semen, vaginal fluid, and teeth) in order to establish biological fluid- and tissue-specific transcriptome (total mRNA) degradation profiles and patterns that may be used to estimate the age of a sample. Whole transcriptome sequencing of RNA isolated from fresh and aged samples (0 days to 360 days old) will be performed to evaluate the patterns of RNA degradation in relation to sample age. Whole transcriptome data will be used to determine the pattern and rate of degradation for each individual mRNA transcript in each sample type. This data will be used to determine the transcripts in each sample type that have degradation patterns and rates most closely correlated with sample age. Preliminary sequencing results are very promising and offer insight as to the identity of the most accurate mRNA markers for determining sample age. The long-range plan of this study is to use the data gained in the full transcriptome analysis of each sample type to develop qPCR assays that can provide the same information with a more simplified technique that is adaptable to crime laboratories. The qPCR assays developed in this study will be used to assess the ability of the identified RNA markers to establish sample age in an increased number of samples from a larger donor pool. ca/ncf