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mRNA-based skin identification for forensic applications.

Visser M, Zubakov D, Ballantyne KN, Kayser M - Int. J. Legal Med. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Forensic Molecular Biology, Erasmus MC University Medical Center Rotterdam, P.O. Box 2040, 3000 CA, Rotterdam, The Netherlands.

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.

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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
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Fig3: 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

Mentions: All three skin-targeted mRNA markers proved to be exceptionally sensitive for the detection of skin cells given the assays applied (Fig. 3). From full thumbprints, all three mRNA markers in all samples except one were detected with a Ct value below 40, with average Ct values of 34.72 for CDSN, 36.38 for LOR, and 36.45 for KRT9 while the average Ct for the reference gene ACTB was 35.8 (primer set ACTB-74). Dropout of KRT9 was observed in one full print, and the donor of this particular print appeared to be a poor shedder based on the expression levels detected in other genes and thumbprints examined. The ΔCt values were generally below the 2.7 threshold as determined in the previously described analysis, with the exception of the LOR ΔCt (3.1) obtained from the poor shedder and the KRT9 ΔCt at 2.8 of one other full thumbprint.Fig. 3


mRNA-based skin identification for forensic applications.

Visser M, Zubakov D, Ballantyne KN, Kayser M - Int. J. Legal Med. (2011)

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
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046345&req=5

Fig3: 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
Mentions: All three skin-targeted mRNA markers proved to be exceptionally sensitive for the detection of skin cells given the assays applied (Fig. 3). From full thumbprints, all three mRNA markers in all samples except one were detected with a Ct value below 40, with average Ct values of 34.72 for CDSN, 36.38 for LOR, and 36.45 for KRT9 while the average Ct for the reference gene ACTB was 35.8 (primer set ACTB-74). Dropout of KRT9 was observed in one full print, and the donor of this particular print appeared to be a poor shedder based on the expression levels detected in other genes and thumbprints examined. The ΔCt values were generally below the 2.7 threshold as determined in the previously described analysis, with the exception of the LOR ΔCt (3.1) obtained from the poor shedder and the KRT9 ΔCt at 2.8 of one other full thumbprint.Fig. 3

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Forensic Molecular Biology, Erasmus MC University Medical Center Rotterdam, P.O. Box 2040, 3000 CA, Rotterdam, The Netherlands.

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.

Show MeSH