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 ((similar to)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.