The author(s) shown below used Federal funding provided by the U.S. Department of Justice to prepare the following resource: Document Title: Identification of Forensically Relevant Fluids and Tissues by Small RNA Profiling Author(s):

The serology-based methods routinely used in forensic casework for the identification of biological fluids are costly in terms of time and sample and have varying degrees of sensitivity and specificity. The use of messenger RNA (mRNA) profiling (a molecular genetics based approach) has been proposed to supplant conventional methods for body fluid identification. However, the size of the amplification products used in these mRNA assays (~100-250 nt) may not be ideal for use with degraded or compromised samples frequently encountered in forensic casework. There has been an explosion of interest in a class of small non-coding RNAs, microRNAs (~20-25 bases in length), with numerous published studies reporting that some miRNAs are expressed in a tissue specific manner. Additionally, another novel class of small regulatory RNAs, piwi-interacting RNAs (~26-30 bases in length), were initially identified only in germ-line cells (including sperm-producing cells) in mammals. We therefore theorized that it may be possible that this class of small RNAs could be ideally suited for the identification of semen. Through the current study, we sought to: 1) determine whether novel body fluid and tissue specific small RNAs, such as microRNAs or piRNAs, can be identified that would permit the identification of forensically relevant biological fluids and tissues (i.e. blood, semen, saliva, vaginal secretions, menstrual blood and skin), 2) demonstrate the suitability of the small RNA profiling assays for use with forensic casework (performance with low template samples, admixed body fluid samples, and also mock casework samples), and 3) compare other body fluid identification strategies (mainly mRNA profiling) in order to determine the most suitable strategy for the analysis of environmentally impacted or degraded samples frequently encountered in forensic casework. We performed an extensive comprehensive screening of ~1,200 microRNAs (through miRBase v.16) and have identified a panel of 18 differentially expressed miRNAs that permit the identification of all forensically relevant body fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). An initial validation of assays incorporating these miRNA biomarkers was performed which included an evaluation of specificity, sensitivity, and performance with admixed body fluid samples. We compared the ability to accurately identify the body fluid of origin of compromised or environmentally impacted body fluid samples using miRNA and mRNA profiling. The analysis (i.e. determination of the body fluid of origin) of the validation samples was aided by a novel statistical approach we developed for the analysis of normalized miRNA express data involving the use of logistic regression models. This permits an objective “prediction” of the presence of the above-mentioned body fluids and tissues using a multi-candidate approach rather than the two-candidate approach previously used. While at this time it cannot be conclusively determined whether miRNA profiling is a superior to mRNA profiling for the analysis of environmentally impacted samples, the results of our work demonstrate the ability to successful use a miRNA profiling approach for the identification of the body fluid or tissue origin of forensic biological evidence with picogram-level sensitivity. This resource was prepared by the author(s) using Federal funds provided by the U.S. Department of Justice. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. 3 ____________________________________________________________________________________________