Practical evaluation of an RNA-based saliva identification method

Identifying saliva in samples found at crime scenes is important to clarify the tissue origin of DNA obtained for identification of individuals. Recently, a novel messenger RNA-based approach using two saliva-specific markers, Statherin (STATH) and Histatin 3 (HTN3), has been reported. This method can identify saliva more specifically than conventional amylase-based methods. Here, we performed several evaluations related to applying this method to real-world forensic work. First, we evaluated the effects of exposure to blue light (450nm) or to the reagent on Phadebas paper, which are direct methods used to locate saliva stains, on the stability of the RNA markers. The results demonstrate that exposure to the two direct tests did not affect the stability of the RNA markers. Second, we performed a comparative analysis of RNA-based and amylase-based conventional methods to examine the sensitivity and stability of the markers under various storage conditions. Although there was no difference in the sensitivity of the two methods for detecting 1-day-old saliva stains, a time-course study demonstrated that the RNA saliva markers were less stable than amylase, especially in wet conditions. During this time-course experiment, the stability of human DNA was also investigated. Although DNA was also unstable in wet conditions, it was more stable than the RNA markers in dry conditions. Taking the above results into consideration, we suggest that the RNA method could be introduced to current saliva identification procedures and should be used as a supplementary method to strongly support identification of saliva by the amylase-based method.