Developmental validation of a miRNA panel for forensic body fluid identification

As submitted by the proposer:

MicroRNAs (miRNAs) are small non-coding RNAs 18-25 nucleotides in length that have been identified and evaluated as potential markers for the identification of forensically relevant body fluids. There is significant interest in the use of miRNAs for forensic casework because of their short length and high resistance to degradation, potentially allowing for robust detection in degraded samples. They have also been shown to co-extract and be detectable in DNA extracts, which could make the use of miRNAs a more streamlined and easily implementable molecular body fluid identification method than other described methods.

This proposed work is a continuation of a previously NIJ-funded project utilizing high-throughput sequencing (HTS) of eight forensically relevant biological fluids to identify microRNAs with tissue-specific expression. Through that work, candidate microRNAs were developed and expression patterns in the eight biological fluids assessed, ultimately identifying eight microRNAs, including a pair of endogenous reference miRNAs that allow for normalization of expression without evaluation of the RNA or known input quantity. This panel uses expression detected using reverse-transcription quantitative PCR (RT-qPCR) to identify and differentiate feces, urine, peripheral blood, menstrual secretions, semen, and saliva. Identification of the biological fluids was found to be reliable across population samples of mixed ages, ethnicities, and gender (where appropriate), and detectable at picogram-level RNA
quantities.

We propose to build on previous work by evaluating markers for vaginal secretions and perspiration, and evaluate the final panel for identification success in mixed samples. An analysis method will be developed for accurate identification with confidence intervals established for each body fluid. We will follow this by performing a comparative analysis between analysis methodologies, a study acutely needed to address performance differences observed in the literature.

Using the platform that delivers optimal performance, final characterization of the optimized panel of microRNAs will be conducted. An expansion on the population study previously performed will provide greater understanding of population variations. Panel performance for body fluid identification of miRNAs in DNA extractions will be assessed and compared to paired RNA extracts. Detection in compromised samples, limit of detection, and species specificity will be evaluated according to developmental validation guidelines. Finally, reproducibility will be assessed, both in-lab and by sending blinded samples, reagents and protocols to external laboratories for concordance testing. Upon completion of this work, this microRNA panel has the potential for rapid and inclusive discrimination of the body fluids encountered in forensic evidence, and capability for rapid implementation.

Note: This project contains a research and/or development component, as defined in applicable law.

ca/ncf