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WIREs Forensic Sci

RNA‐based approaches for body fluid identification in forensic science

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Abstract In forensic science, the identification of body fluids present in a stain can aid in the reconstruction of crime scenes and support activity‐level information in some cases. RNA‐based methods for body fluid identification address some limitations associated with conventional testing, including a lack of specificity and sample destruction. mRNA profiling by endpoint reverse transcription polymerase chain reaction (RT‐PCR) is a confirmatory RNA method currently used by some forensic laboratories in casework due to the availability of capillary electrophoresis instruments. This method is highly specific and reliable when the RNA input is within the optimal range. When RNA input is below the limit of detection or too high, then false negatives and false positives/cross reactivity can occur, respectively. Real‐time quantitative PCR (qPCR) is quantitative, more sensitive, and efficient than endpoint PCR, and does not require post‐PCR processing. A reverse transcription (RT) step may also be included in qPCR (RT‐qPCR), enabling mRNA quantification. However, there are concerns regarding the lack of consistency and standardization of RT‐qPCR methodology and reporting of results. Additionally, the number of currently available fluorophores somewhat reduces multiplexing ability. More recently, approaches involving Piwi‐interacting biomarkers, NanoString barcodes, and RNA sequencing have been developed. This review will cover the current and developing RNA‐based techniques for forensic body fluid identification, focusing on endpoint and quantitative RT‐PCR using mRNA. The advantages and limitations of these along with alternative methods will be discussed, and how they may be further developed to advance forensic body fluid identification. This article is categorized under: Forensic Biology > Body Fluid Identification Forensic Biology > Applications of RNA
Capillary electrophoresis allows for separation of RT‐PCR products by size, enabling detection
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Multiplexing is possible with RT‐qPCR and HRM analysis, as amplicons have different melting temperatures (Tm). Melt curves are generated for each amplicon (left). The difference in melt curves between the targets (green and orange) and reference (blue line) are plotted to produce a difference curve (right)
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The normalization of RT‐qPCR data is commonly achieved using a reference gene. The difference between the two Ct values at a set threshold of fluorescence is the delta Ct (dCt)
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Diagram of RT‐qPCR. The process begins with RNA extraction, separate cDNA synthesis and amplification for two‐step or cDNA synthesis and amplification by PCR in the same tube for one‐step, followed by analysis
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When a threshold is set, Ct values produced from a dilution series of RNA may be plotted against the input concentration or dilution factor to obtain reaction sensitivity and efficiency
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With increasing PCR cycles, the quantity of PCR products (and therefore fluorescence) increases exponentially until it reaches a linear phase, then plateaus. RT‐qPCR measures fluorescence in the exponential phase, whereas endpoint RT‐PCR measures during the plateau
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General process of mRNA‐based RT‐PCR for body fluid identification. The workflow begins with sampling of the stain, RNA extraction, complementary DNA synthesis, PCR amplification, capillary electrophoresis to separate and detect the body fluid specific amplicons based on size, and analysis of the results
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Forensic Biology > Applications of RNA
Forensic Biology > Body Fluid Identification

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