MODERN PERSPECTIVES IN MOLECULAR IDENTIFICATION OF FORENSIC BODY FLUIDS: A COMPREHENSIVE REVIEW OF MRNA EXPRESSION ANALYSIS VIA RT-QPCR.

Abstract

The identification of forensic body fluids plays a critical role in reconstructing crime scenes and providing contextual evidence in criminal investigations. Traditional biochemical and serological methods, while useful for preliminary detection, often suffer from limitations in specificity, sensitivity, and reliability, particularly when dealing with degraded or mixed samples. In recent years, molecular approaches—especially messenger RNA (mRNA) expression analysis using reverse transcription quantitative polymerase chain reaction (RT-qPCR)—have emerged as powerful alternatives for accurate body fluid identification. This review provides a comprehensive overview of modern perspectives in mRNA-based forensic analysis, focusing on the biological basis of tissue-specific gene expression, the principles and workflow of RT-qPCR, and recent advancements in marker discovery and assay development. Specific mRNA markers associated with commonly encountered body fluids, including blood, semen, saliva, vaginal fluid, and menstrual blood, are discussed in detail, highlighting their diagnostic potential and forensic applicability. The review also examines the impact of environmental factors on RNA stability and outlines strategies to mitigate degradation, such as optimized extraction methods and short amplicon design. Furthermore, recent developments in multiplex assays, computational analysis, and machine learning approaches are evaluated for their role in improving accuracy and interpretation, particularly in complex and mixed samples. Validation studies and casework applications are also considered, demonstrating the growing acceptance and reliability of mRNA-based techniques in forensic practice. Despite ongoing challenges related to standardization and legal admissibility, the integration of mRNA profiling with emerging biomarkers such as microRNA and epigenetic markers offers promising future directions. Overall, mRNA expression analysis via RT-qPCR represents a significant advancement in forensic molecular biology, enhancing the specificity, sensitivity, and evidentiary value of body fluid identification and contributing to more accurate and scientifically robust criminal investigations.