Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of timewise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissuespecific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice.

In forensic science, the unequivocal identification of the cellular origin of crime scene samples used for DNA profiling can provide crucial information for crime scene reconstruction. We have previously shown that various mRNA markers from genes with expression patterns specific for blood and saliva can be established from whole-genome expression analysis of time-wise degraded samples and were stable enough to specifically identify blood and saliva stains up to 180 days of age. Here, we showed that nine blood-specific and five saliva-specific mRNA markers can be amplified successfully and reliably in much older blood (13-16 years) and saliva (2-6 years) stains, respectively, suggesting their suitability for tissue identification in forensic case work. Moreover, our findings imply that forensic RNA testing can be reliable and robust if degraded samples are considered in the marker ascertainment procedure, with promising expectations beyond tissue identification purposes.