Hyperplex PCR for forensic mRNA-based cell typing

Identification of body fluids or cell types may strengthen the evidential value of biological crime scene stains by clarifying the connection between the evidence and a specific activity. Studying gene expression through mRNA analysis is an effective way to determine the cell type, due to the specific expression of some genes in certain cells. A method that allows for quick analysis of high numbers of mRNA markers spanning all relevant cell types and has a large dynamic range is therefore warranted. Hyperplex PCR (hpPCR) is a new technique that enables simultaneous detection of up to 100 RNA or DNA targets. Here, we describe the development and evaluation of a proof-of-concept hpPCR 17-plex mRNA assay for forensic cell type determination. The targets and primer pairs were chosen from the capillary electrophoresis (CE) based CellTyper method. The hpPCR assay includes markers for blood, menstrual blood, vaginal mucosa, saliva, nasal secretion and semen. Samples of all these cell types, plus mixtures thereof, were analyzed in two forensic laboratories. The hpPCR mRNA assay detected all the expected markers for semen, blood, saliva, nasal secretion and vaginal secretion. CellTyper showed similar results but gave partial detection for vaginal secretion. Overall, the hpPCR gave equivalent or enhanced detection of the expected markers compared to CellTyper. In mixtures, hpPCR showed somewhat improved sensitivity over CellTyper for minority contributions of semen and equal detection for vaginal secretion compared to CellTyper. In conclusion, hpPCR provides a promising alternative to capillary electrophoresis and sequencing for mRNA-based cell type identification. Further development of the hpPCR assay may include adding more mRNA markers, for the existing cell types or new ones, as well as RNA-SNPs to connect the mRNA result to an individual.