Forensic MicroRNA Analysis of Body Fluids Relating to Sexual Assaults

DNA profiling has become a universal technique
for identifying individuals for evidential use in courts of law. In more complex cases such as sexual assaults, identifying the origin of a stain or sample offers valuable information as to the events that occurred. Currently, many ‘in service’ body fluid identification (BFID) techniques are presumptive, require significant sample volumes and generate false positives. As such, the development of a highly specific and reliable BFID technique would be highly beneficial to forensic scientists in provide more informative and reliable evidence.

MicroRNAs (miRNA) are short, stable, non-coding RNA’s which modulate gene expression. Expression of some of these miRNA are body fluid specific, making them a potentially robust tool for BFID. The possibility for the integration of a robust, miRNA based BFID technology for forensic casework employing stem-loop reverse transcription and qPCR analysis was the theme of the research presented here. To be incorporated into the workflow of current forensic laboratories, the protocol must be able to be carried out alongside current techniques with limited addition of cost, equipment, analysts and time.

A range of custom designed miRNA markers were
analysed on vaginal material, menstrual blood, saliva, venous blood, semen, seminal fluid and skin. Screening indicated specificity of hsa mir-124 to vaginal material, hsa-mir-10a, 135a and 888 to semen, hsa-mir-412 and 507 to menstrual blood, hsa-mir-144-3, 144-5, 142 and 451 to blood and although highly expressed in saliva, hsa-mir-205 was also observed in vaginal material. Universal expression was observed in hsa-mir-93, 508, 1260b and SNORD 47 providing a means of normalisation through the designation of these markers as endogenous controls. A combined panel of markers are presented which were capable of identifying all body fluids, excluding skin from single stains. The panel was successful at identifying certain mixtures such as semen within vaginal material but was unable to confirm saliva presence within vaginal material. Screening of hsa-mir 205 within vaginal material uncovered the observation that hsa-mir-205 was impacted by the use of female contraception.

Once a full BFID panel was generated the robustness of the markers was further analysed across the menstrual cycle. No significant difference (p>0.001) was seen in markers highly expressed in vaginal material during screening (hsa-mir-124, 203a, 205). Expression of non specific markers highlighted the importance of the optimisation of input miRNA. Differential extraction of genetic material was found to be detrimental to miRNA sample integrity. As such, total DNA extraction was employed for vaginal swabs obtained from volunteers following unprotected sexual intercourse, markers hsa mir-10a, 135a and 888 were able to successfully detect semen presence for up to 96 hours.

The data generated to date has highlighted a number of miRNA markers that provide a platform for BFID. The developed protocol is reliable and robust; requiring minimal optimisation and is capable of integration with current laboratory workflow with minimum implications to time and cost. The markers identified for BFID have been implemented within studies that are representative of real case scenarios, and have demonstrated their ability to be stable, specific and successful in the identification of certain body fluids. Overall, this research showcases a reliable and body fluid specific protocol capable of being performed alongside DNA profiling.