Research On The Application Of MicroRNA In Forensic Body Fluid Identification

Objective:The identification of biological stains and tissues?Peripheral blood,saliva,semen,and menstrual blood?can provide crucial evidence for crime scene reconstruction and connect a suspect to a crime scene.MicroRNA?mi RNA?are regarded as a potential biomarker for forensic body fluid identification because of the tissue-specific expression pattern.MicroRNA are also stable against degradation.The objective of this study is to screen a panel of tissue specific miRNA and develop a detection and aglrithm to determine the tissue origin of forensic samples.Methods:This study describes a complete and reliable MicroRNA workflow solution encompassing the selection of Tissue-Specific MicroRNA Markers,the comparation of RNA extraction kits,the screening of Real Time PCR methods,the design adjustment of the primers,the optimization of the amplification system and the reaction conditions.The system were evaluated by authentic specimens,all assays were run in triplicates on QuantStudio^TM7 platform.RNU6b was used as the reference gene to normalize the target MicroRNA to calculate the Delta Ct values?Delta Ct was calculated by subtracting the Ct value of the RNU6b from the Ct value of the target MicroRNA?.Amplification signals were computed with the QuantStudio^TM7Real-Time PCR Software V1.3?Thermo Fisher Scientific,USA?.Relative quantification of MicroRNA expression was performed by the2^??Ct.Origin 8.0 was used to analyze significant differences between the body fluids.Principal components analysis?PCA?were performed by the Rv3.2.3,and the plot was created by R package ggplot2.The amplification system was validated by stability,sensitivity and the comparation results with BGI Genomics Technology Co Ltd and Thermo Fisher Scientific.Results:On the basis of a large number of experimental studies,MicroRNA extraction method was established successfully based on the MiRNeasy?Mini Kit.The MicroRNA yield and extraction efficiency are both high enogh for the following detection work.The OD260/280 values of all RNA samples were about 2.0 which were suitable for array detection.Isolation method efficiency was measured through RT-qPCR analysis.All the markers showed different expression pattern among different body fluids,miR-451a and miR-144-3p could be used to distinguish blood from non-blood.Menstrual blood or peripheral blood could be identified through miR-214-3p.miR-888-5p and miR-891a-5p was highly expressed in semen.miR-205-5p was highly expressed in menstrual blood.The Principal component analysis?PCA?revealed that samples belonging to the same body fluid tend to cluster together and that overall samples display distinct MicroRNA expression signatures.The component 1 and 2 could explain the total variance of 84.5%.Trace biological evidence is very common in the criminal scene,so assays need to be sensitive enough to accomadate the forensic evidience.The system demonstrated a good stability and sensitivity to different sample types.We have got a concordant results with BGI Genomics Technology Co Ltd and Thermo Fisher Scientific.Conclusion:In this study,we have selected a panel of MicroRNA markers to identify Peripheral blood,saliva,semen,and menstrual blood.MicroRNA extraction method have been constructed with good performance in quantity and quality for forensic evidence.A stable MicroRNA detection system and a novel statistics were also been developed to identify different tissue types.Validation study showed that the system is stable and sensitive.So the method is a promising tool for body fluid and tissue identification.