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Developmental validation of an mRNA kit: A 5-dye multiplex assay designed for body-fluid identification
Yuanyuan Xiao1, Mengyu Tan1, Jinlong Song1
1Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, PR China.
Abstract:
Identifying the sources of biosamples found at crime scenes is crucial for forensic investigations. Among the markers used for body fluid identification (BFI), mRNA has emerged as a well-studied marker because of its high specificity and remarkable stability. Despite this potential, commercially available mRNA kits specifically designed for BFI are lacking. Therefore, we developed an mRNA kit that includes 21 specific mRNA markers of body fluids, along with three housekeeping genes for BFI, to identify four forensic-relevant fluids (blood, semen, saliva, and vaginal fluids). In this study, we tested 451 single-body-fluid samples, validated the universality of the mRNA kit, and obtained a gene expression profile. We performed the validation studies in triplicates and determined the sensitivity, specificity, stability, precision, and repeatability of the mRNA kit. The sensitivity of the kit was found to be 0.1 ng. Our validation process involved the examination of 59 RNA mixtures, 60 body fluids mixtures, and 20 casework samples, which further established the reliability of the kit. Furthermore, we constructed five classifiers that can handle single-body fluids and mixtures using this kit. The classifiers output possibility values and identify the specific body fluids of interest. Our results showed the reliability and suitability of the BFI kit, and the Random Forest classifier performed the best, with 94% precision. In conclusion, we developed an mRNA kit for BFI which can be a promising tool for forensic practice.
Insights
Forensic investigations benefit from a new mRNA kit for body fluid identification (BFI). This tool accurately identifies blood, semen, saliva, and vaginal fluids, enhancing crime scene analysis.
Area of Science:
- Forensic science
- Molecular biology
- Genetics
Background:
- Accurate identification of biological samples at crime scenes is vital for forensic investigations.
- Messenger RNA (mRNA) offers high specificity and stability as a marker for body fluid identification (BFI).
- A commercial mRNA kit specifically for BFI is currently unavailable.
Purpose of the Study:
- To develop and validate a novel mRNA kit for the identification of four forensic-relevant body fluids: blood, semen, saliva, and vaginal fluids.
- To assess the sensitivity, specificity, stability, precision, and repeatability of the developed mRNA kit.
- To create classifiers for identifying single body fluids and mixtures using the mRNA kit.
Main Methods:
- Development of an mRNA kit comprising 21 specific mRNA markers and 3 housekeeping genes for BFI.
- Testing of 451 single-body-fluid samples and validation studies in triplicates.
- Construction of five classifiers, including Random Forest, to analyze body fluid samples and mixtures.
Main Results:
- The developed mRNA kit demonstrated high reliability and suitability for BFI.
- The kit achieved a sensitivity of 0.1 ng and was validated using various mixtures and casework samples.
- The Random Forest classifier exhibited the highest precision at 94% for identifying body fluids.
Conclusions:
- A novel mRNA kit for body fluid identification has been successfully developed.
- The kit is a promising tool for forensic practice, offering reliable identification of key body fluids.
- The developed classifiers effectively identify single fluids and mixtures, with Random Forest showing superior performance.
