Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

Abstract In the present work, we have evaluated eight reportedly blood-specific mRNA markers (HBB, HBA, ALAS2, CD3G, ANK1, PBGD, SPTB, AQP9) in an attempt to determine the most suitable ones for use in forensic applications based on their sensitivities, specificities and performance with casework samples. While varying levels of expression were observed, all markers were relatively sensitive requiring as little as 1 ng of RNA input into the reverse transcription (RT) reaction. In singleplex reactions, seven of the eight analyzed blood markers (all except AQP9) demonstrated a high degree of specificity for blood. In multiplex reactions, non-reproducible cross-reactivity was observed for several of the mRNA markers, which was reduced and, in most cases, eliminated when less input total RNA was used. Additionally, some cross-reactivity was observed with tissue and animal samples. Despite differences in the observed sensitivity and specificity of the blood markers examined in this study, a number of the candidates appear to be suitable for inclusion in appropriately validated multiplex mRNA-based body fluid identification systems.