Considering the effect of stem-loop reverse transcription and quantitative PCR analysis of blood and saliva specific microRNA markers upon mixed body fluid stains

Forensic RNA analysis is gathering pace with reports of messenger RNA analysis being used in case work, and with microRNA being increasingly researched. Such techniques address a fundamental issue in body fluid identification, namely increased specificity over existing chemical tests, and the incorporation of additional body fluids such as vaginal material. The use of RNA analysis will be of particular value to sex offences, where there can be a mixture of multiple body fluids from different people. The aim of this study was to determine whether microRNA based body fluid identification tests can be applied to mixed body fluid samples.

Blood and saliva were acquired from volunteers and underwent total RNA extraction. Mixed samples were prepared using a range of ratios from 1:1 to 10:1. Each mixed sample then underwent a blood–saliva differentiation test developed in-house, which includes stem-loop reverse transcription and real-time PCR analysis. Aliquots following mixture preparation also underwent standard STR analysis, utilising Quantiplex and Next Generation Multiplex kits.

Data relating to the development of an in-house blood–saliva differentiation test is presented, in which it has been demonstrated that such a test has a lower limit of detection than the enzymatic equivalent.

It has been shown that not only is it possible to determine the presence of more than one body fluid, it is also possible to determine the major body fluid contributor as well as the minor contributor.