Forensic casework analysis often relies on DNA-based techniques, yet RNAbased methodologies such as Fluorescence in situ hybridization (FISH) present valuable complementary tools for deciphering crime scene biology. FISH employs fluorescent probes to bind specific RNA sequences, offering insights into gene expression, mRNA processing, and cellular localization within biological evidence.
This technique is particularly adept at characterizing diverse cell types present in body fluids and tissues relevant to criminal investigations, thus linking specific cellular origins to pertinent events. Leveraging the molecular signatures of nucleotide sequences (including DNA, miRNAs, and STRs) with fluorescent nucleic acid probes holds promise for elucidating intricate details of crime scene biology. This exploration delves into the essential intricacies and considerations of RNA-FISH profiling in forensic casework analysis, emphasizing its critical applications in deciphering the complex cellular landscapes encountered in criminal investigations.
The central dogma of genetics offers a framework to understand how genetic information aids in analyzing and interpreting genetic evidence in criminal investigations. Over the past 25 years, molecular biology has seen advances in its forensic applications, notably the use of RNA to identify body fluids alongside traditional DNA identification techniques. One significant method is RNA fluorescent in-situ hybridization (RNA-FISH), which visualizes gene expression in tissues without extracting RNA or DNA.
FISH, originally used to target DNA, has been adapted to label RNA, making it valuable for diagnosing diseases, identifying microorganisms, and in forensic casework. Although RNA-FISH has shown promise, it remains underutilized in forensic biology. The technique can potentially revolutionize body fluid identification, which, until now, has relied on conventional methods that are often time-consuming, labor-intensive, and prone to low specificity and sensitivity.
Proteins and messenger RNA (mRNA) are expressed in a tissue-specific manner, which is crucial for definitively identifying the tissue or cell source of biological evidence. As molecular biology advances, targeting mRNA has emerged as a superior option for body fluid identification. The timeline of mRNA profiling studies shows its capability in identifying body fluids such as saliva, blood, semen, and vaginal secretions. Optimized methods that co-isolate mRNA and DNA from dried body fluid stains have resolved the issue of missing contributors when separate samples are taken from mixed stains.
Despite advancements in mRNA profiling, conventional body fluid identification techniques still hold value in certain forensic scenarios. For example, in sexual assault cases, identifying whether blood is menstrual or peripheral can provide critical probative evidence. As Juusola and Ballantyne (2003) argued, bypassing traditional identification tests may increase efficiency, but specific scenarios require body fluid identification for important legal arguments.
Key breakthroughs in mRNA profiling include the identification of candidate tissue-specific genes and the development of multiplex RT-PCR, a method for detecting multiple target genes at once. While multiplex RT-PCR is widely used, RNA-FISH offers another layer of forensic casework by allowing the visualization of RNA molecules in their original context. RNA-FISH uses fluorescently labeled probes to target specific mRNAs, enabling scientists to detect and visualize these transcripts in-situ, within the cells where they are expressed.
A noteworthy method, RNA Suspension-FISH (S-FISH), was developed by Williams et al. (2014) using a locked nucleic acid (LNA) probe for keratin 10 (KRT10) mRNA, a marker for epithelial cells. RNA S-FISH minimizes cell loss while maintaining signal strength, an essential consideration given the limited sample sizes often encountered in forensic investigations.
The application of RNA-FISH is especially advantageous in detecting spatiotemporal expression of specific mRNAs and microRNAs (miRNAs), which can provide clues about the origin and function of biological samples. For example, detecting specific mRNAs in menstrual blood can distinguish it from peripheral blood, providing crucial evidence in certain criminal cases. Additionally, RNA-FISH can detect mRNA transcripts for human cytochrome b and beta-spectrin in blood samples, confirming that the sample is of human origin.
Fluorescence microscopy plays a vital role in RNA-FISH. By selecting specific wavelengths of light to excite fluorophores attached to RNA probes, scientists can visualize the presence of specific RNA molecules. This process relies on exciter filters, beam splitters, and barrier filters to detect the emitted fluorescence, signaling the presence of the targeted RNA in the sample.
Beyond forensic applications, RNA-FISH can also be used to detect biological toxins, aiding investigations into poisoning, homicides, biological warfare, environmental contamination, and food safety. My research on Conus magus venom duct cells and Apis dorsata venom illustrates how RNA-FISH can identify specific venom proteins, highlighting the technique's broader applications.
Despite the potential of RNA-FISH, there is no established system for its robust use in forensic casework. However, existing foundations in mRNA profiling via RT-PCR and DNA extraction methodologies provide a basis for the development of RNA-FISH in identifying body fluids. Advances in RNA-FISH probe design, including optimization for factors like probe length, GC content, and salt concentration, are key to improving its specificity and performance.
Applying RNA-FISH alongside RNA-DNA based technologies offers a more definitive approach to body fluid and tissue identification in forensic investigations. Since body fluids contain multiple cell types, identifying these cells, linking them to a potential donor, and understanding their role in the crime can strengthen forensic evidence. As cellular life can persist for hours or even days after death, RNA-FISH can offer valuable insights even in compromised biological samples, helping to solve crimes and provide justice.
In summary, integrating RNA-FISH with existing forensic techniques holds great promise for more accurate identification of biological evidence in criminal investigations. This approach not only enhances the reliability of forensic evidence but also opens new avenues for the analysis of body fluids, tissues, and potential toxins, providing a critical link between crime scene biology and the resolution of complex cases.