mRNA profiling using a minimum of five mRNA markers per body fluid and a novel scoring method for body fluid identification

Messenger RNA (mRNA) expression varies among cell types; therefore, analyses for the presence of particular mRNAs can be used to identify biological fluids in forensic samples. For this work, several novel markers were characterized for saliva, cervicovaginal fluid (CVF), blood, and menstrual blood (MB). The new markers were used in combination with previously described markers to develop four multiplex polymerase chain reaction assays. These multiplexes incorporate two housekeeping and a minimum of five markers for each of the following forensically relevant body fluids: semen, saliva, CVF, blood, and MB. A large number of samples (> 200) were analyzed to determine specificity of each marker. The majority of the markers were detected at low frequencies in non-target body fluids. Because markers were not specific to their respective target body fluids, a scoring system was developed to minimize the chances of misidentification of a sample due to marker expression in a non-target body fluid. Each marker was given a numerical value related to its correct (target body fluid) versus incorrect (non-target body fluid) expression in samples of known origin. For each of the five body fluids, the marker values of those mRNA markers that were present in a sample were added to produce a body fluid score. Threshold scores were then determined for the identification of each body fluid. Although this study highlights the complexity of body fluid identification, particularly in differentiating blood and MB, the use of threshold scores allowed for reliable body fluid identification in the samples tested.