Abstract:
In forensic science, the determination of the tissue source of a biological fluid can often provide important information that can increase the evidential value of a sample. While conventional methods for body fluid identification are often presumptive and lack specificity, messenger RNA (mRNA) profiling can act as a confirmatory test with higher specificity than most conventional tests. Due to its tendency to cause cross-reactions within developed mRNA assays, this research aimed to identify an mRNA marker capable of detecting the presence of nasal mucosa. Whole transcriptome sequencing and bioinformatic analysis techniques were used to identify differentially expressed genes within nasal samples. An assessment of the function and predicted tissue distribution of these genes helped to narrow the pool down to five candidate markers; KCNQ1OT1, GBP5, PTPRC, OPRPN, and TLR1. A sixth candidate marker, BPIFA1, was obtained from the literature. The sequencing data also supported the body fluid specificity of CellTyper 2 markers, and the differential expression patterns corresponded with the cross-reactions observed for nasal mucosa. Primers were designed for each of the six candidate nasal markers, and an additional set for BPIFA1 was obtained from the literature for comparison. During sensitivity testing all seven primer sets successfully amplified their target within nasal samples. Following this, specificity testing highlighted the two most promising candidate markers. Further testing uncovered variations in the amplification of the top two markers within nasal samples of different origins (illness, allergy, eating spicy food, common/unknown). This testing suggested that the quality, quantity, and type of nasal sample is likely to influence marker detection. Additional testing may be required to inform decisions around the inclusion of each marker and the optimal primer concentration for forensic type samples. The origin of nasal samples also caused variations in the occurrence of cross-reactions with CellTyper 2 markers, highlighting the potential for interpretation issues during analysis. Testing was carried out to assess the performance of each marker in duplex and when multiplexed with the saliva markers from CellTyper 2. The results suggest that the multiplexing of nasal and saliva markers is unlikely to affect the detection of either fluid. Therefore, in order to counteract possible interpretation issues, the inclusion of nasal markers within CellTyper 2 may be the most beneficial way to implement testing for nasal mucosa.