mRNA-based skin identification for forensic applications

Abstract

Although the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in forensic analyses. We examined 11 candidate genes with skin-specific expression, as ascertained from expression databases and the literature, as well as five candidate reference genes ascertained from previous studies, in skin samples and in other forensically relevant tissues. We identified mRNA transcripts from three genes CDSN, LOR and KRT9, showing strong over-expression in skin samples relative to samples from forensic body fluids, making them suitable markers for skin identification. Out of the candidate reference genes tested, only ACTB showed similarly high expression in skin and body-fluid samples, providing a suitable reference marker for quantitative real-time PCR (qPCR) analysis of skin. Analyses of palmar and thumbprint skin samples indicate that our qPCR approach for the three skin-targeted mRNA markers, as well as the reference mRNA marker ACTB, is highly sensitive, allowing successful detection of minute amounts of skin material including full, half and quarter thumbprints, albeit with decreased success in decreasing print material. Furthermore, thumbprints stored for 6.5 months provided similar results relative to freshly analysed samples implying reasonable time-wise stability of the three skin-targeted mRNAs as well as the ACTB reference mRNA. Our study represents the first attempt towards reliable mRNA-based skin identification in forensic applications with particular relevance for future trace/touched object analyses in forensic case work. Although the approach for skin identification introduced here can be informative when applied on its own, we recommend for increased reliability the integration of (one or more of) the skin-targeted mRNA markers presented here into multiplex assays additionally including mRNA markers targeting alternative cell types expected in forensic samples.

Fig. 1

Ct values of the reference gene ACTB from biopsied skin samples and those from forensically relevant body fluids. Equal amounts of total RNA (100 ng) were used for cDNA synthesis in each sample. Error bars represent the maximum and minimum observed Ct values from two to four individuals for each sample type

Fig. 2

ΔCt values for three skin-targeted mRNA markers CDSN, KRT9 and LOR in various skin samples and across forensically relevant body fluids: Palmar skin (L), n = 15; Palmar skin (R), n = 15; Blood (fingerprick), n = 15; blood (menstrual), n = 3; blood (venous), n = 12; saliva, n = 15; semen, n = 2; vaginal, n = 7. The box for each gene represents the interquartile range (25–75th percentile), and the line within this box is the median value. Bottom and top bars indicate the minimum and maximum value of all the data, respectively, or 1.5 times the interquartile range of the data, whichever is smaller. Outlier values are indicated by closed circles. Note the significantly lower ΔCt values observed in skin compared to non-skin samples. An asterisk indicates that the target gene was not detected in that tissue

Fig. 3

Detection sensitivity of qPCR assays for three skin-targeted mRNA markers CDSN, KRT9 and LOR, with full (n = 5), half (n = 5) and quarter (n = 10) thumbprints. All sample points below the line would be correctly identified as originating from skin, when applying a threshold of 2.7 (see specificity results for further details). An asterisk indicates that dropout was observed, with the number specified for each marker and fingerprint size

Fig. 4

Time-wise stability of three skin-targeted mRNA markers CDSN, KRT9 and LOR after 6.5 months of thumbprint storage. No significant differences were observed between freshly analysed samples (n = 5) and thumbprints stored for 6.5 months at room temperature (n = 10) for all three mRNA markers