Deep Sequencing for Identification and Characterization of MicroRNAs in Forensically Important Biological Fluids

Molecular methods for forensic body fluid identification have yet to achieve widespread acceptance and implementation, particularly due to perceived and actual limitations of the species being detected. The field of investigation around microRNAs or miRs has sufficiently developed that we can begin to consider evaluating miRs for forensic body fluid identification. miRs hold several distinct advantages over mRNA or proteins as a potential molecular method for body fluid ID: they are small in size (19-23 nucleotides long) and without the degradation enhancement sites present in both immature and mature mRNAs (poly-A tail, binding sites on the 5'- and 3'-UTRs). Studies have shown that miRs can be subjected to particularly harsh conditions, including very high and low pH, boiling, freeze/thaw cycles, and formalin-fixing, and still be detectable. They also require much less sample to be detected than mRNA analysis requires and have been shown to be stable in forensic-type samples, stored dried for over 1 year. A handful of studies have investigated miRs for forensic analysis; however, they utilized hybridization assays, which only assay the known miRs in the human transcriptome. This is by no means comprehensive; as of the most recent study, only approximately 700 miRs were known and included in the assay. It is believed that there are over 1000 undiscovered miRs in the human genome, and thus the picture painted by these authors is not a complete one. Additionally, 2 forensically relevant body fluids have never been analyzed for the presence of miRs, and thus the presence of miRs unique to those body fluids has not yet been assessed. We propose to analyze the 8 common forensically-relevant body fluids for the presence of both novel and known miRs using deep sequencing. This next generation sequencing method sequences all transcripts within a sample in parallel, and thus gives a comprehensive analysis of the tissue being sampled. Thus, both already described miRs, as well as any novel miRs not yet described, will be detected if present in that body fluid. We will confirm and characterize any novel miRs discovered through quantitative Real-Time PCR. miRs found to be unique for one body fluid, whether they be novel or already described, will be further characterized using qPCR methods. miRs commonly seen in all tissues will be evaluated for potential use as a control or normalization miR. Species and body fluid specificity will be confirmed by using an organ/tissue panel, population and animal samples. Population samples of each body fluid will be analyzed for characterization across gender (if appropriate), ethnicity, and age, and will also enable comparisons of relative abundance and detection thresholds of the miRs in question. Samples within the same individuals over a short period of time will assess detectability of miRs throughout the menstrual cycle (for vaginal secretions and menstrual blood) or dietary and hydration changes (for saliva, urine, and feces). Limit of Detection will also be evaluated, in order to determine the concentration of RNA necessary to still detect miRs (sensitivity study). In summary, this study has the potential to identify novel, body-fluid specific miRs, to settle the inconsistencies seen in the previous array studies, and begin to characterize miRs according to forensic developmental validation guidelines. ca/ncf