The potential use of Piwi-interacting RNA biomarkers in forensic body fluid identification: A proof-of-principle study.

In the forensic community, RNA profiling has been investigated as a potential method to identify body fluids. Several RNA molecules, including messenger RNA (mRNA), microRNA (miRNA) and circular RNA (circRNA), have been explored as biomarkers to distinguish different body fluids and have led to considerable interest in the development of RNA biomarkers for forensic purposes. Piwi-interacting RNA (piRNA), a class of noncoding RNAs, is a potential biomarker for body fluid identification because of its short length (˜24-32 nt) and specific expression pattern in human tissues. In this proof-of-principle study, we examined the expression levels of four carefully selected piRNAs in forensically relevant biological fluids (venous blood, saliva, semen, menstrual blood and vaginal secretions) using TaqMan quantitative real-time polymerase chain reaction (TaqMan qPCR). piR-55521, which was not detectable in saliva, can differentiate semen from other body fluids because it was strongly expressed in semen compared to the remaining three fluids (> 4000-fold change). Furthermore, piR-55521 could be detected in semen samples made from as little as 200 pg of total RNA, and addition of female component had no effect on the detection limit. Furthermore, the expression differences of other piRNAs, piR-61648, piR-43994 and piR-33151, were statistically significant between at least two types of body fluids. Stability tests also indicated that these piRNAs could be effectively detected in dried samples under laboratory and outdoor conditions for at least six months. Although limited to four piRNAs, this study suggests that the expression pattern of piRNAs could be used to identify body fluids, and that piRNA (piR-55521) is specifically expressed in semen. Such findings suggest that additional work could identify other piRNAs that could serve as biomarkers to identify body fluids.

Expression profile analysis of piwi-interacting RNA in forensically relevant biological fluids.

During the last decade, RNA profiling has emerged as one of the fastest developed methods for discriminating forensically relevant biological samples. As a category of small non-coding RNA, piwi-interacting RNA (piRNA) has recently been proposed to be differentially expressed in different types of body fluids, which indicates that its potential in forensic science is worth exploring. In this study, small RNA from 6 types of biological samples (venous blood, menstrual blood, saliva, semen, vaginal secretions and skin) was prepared and sequenced in order to characterize the expression pattern of piRNA using Ion S5 XL platform. Multiple bioinformatic methods were applied to make interpretation of the massively parallel sequencing data and identify representative biomarkers. A total of 376 piRNAs were initially identified after normalization and filtering. Hierarchical clustering and partial least squares-discriminant analysis (PLS-DA) revealed that their expression profiles exhibited an acceptable discriminating ability for most biological samples. Besides, a panel consists of 37 piRNA candidates was subsequently established for further analysis. The results suggested that with the optimal number of PLS components, the marker-reduced panel was sufficient to construct a PLS-DA model with the same performance as that can be achieved by the entire 376 piRNAs (classification error rate = 0.04). In addition, 5 targeted candidates were further selected for validation. TaqMan RT-qPCR assay results verified the potential of 3 piRNAs (piR-hsa-27622, piR-hsa-1207 and piR-hsa-27493) in distinguishing venous blood and menstrual blood, as well as 2 piRNA (piR-hsa-27493 and piR-hsa-26591) for the discrimination of saliva and vaginal secretions, which emphasized the feasibility of our biomarker selection approach. In brief, our study expanded the amount of potential piRNA biomarkers and demonstrated that the expression features of piRNA could provide valuable information for discriminating forensically relevant biological samples.