Evaluation and Verification of a microRNA Panel Using Quadratic Discriminant Analysis for the Classification of Human Body Fluids in DNA Extracts

Abstract

There is significant interest in the use of miRNA analysis for forensic body fluid identification. Demonstrated co-extraction and detection in DNA extracts could make the use of miRNAs a more streamlined molecular body fluid identification method than other RNA-based methods. We previously reported a reverse transcription-quantitative PCR (RT-qPCR) panel of eight miRNAs that classified venous and menstrual blood, feces, urine, saliva, semen, and vaginal secretions using a quadratic discriminant analysis (QDA) model with 93% accuracy in RNA extracts. Herein, miRNA expression in DNA extracts from 50 donors of each body fluid were tested using the model. Initially, a classification rate of 87% was obtained, which increased to 92% when three additional miRNAs were added. Body fluid identification was found to be reliable across population samples of mixed ages, ethnicities, and sex, with 72–98% of the unknown samples classifying correctly. The model was then tested against compromised samples and over biological cycles, where classification accuracy varied, depending on the body fluid. In conclusion, we demonstrated the ability to classify body fluids using miRNA expression from DNA extracts, eliminating the need for RNA extraction, greatly reducing evidentiary sample consumption and processing time in forensic laboratories, but acknowledge that compromised semen and saliva samples can fail to classify properly, and mixed sample classification remains untested and may have limitations.

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids

“…Therefore, it is proposed in this study to expand the previously reported microbial 16S triplex to incorporate a miRNA reverse transcription‐qPCR (RT‐qPCR) duplex that can now differentiate between vaginal/menstrual secretions, feces, saliva, blood, and semen at the quantification step of the forensic DNA workflow [17]. Our previous work on body fluid classification using miRNAs had previously identified and validated miR‐891a and let‐7g as being useful as markers for distinguishing blood and semen, and as an internal control, respectively [46].…”