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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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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
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Fig1: 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

Mentions: In addition, five commonly used reference genes (ACTB, B2M, GAPDH, PPIB and UBC) [4, 5, 7, 8, 23–26] were tested in skin samples. Of those, the expression of ACTB appeared to be most abundant in skin material (data not shown). When further tested in the various forensically relevant body fluids, ACTB showed similarly high levels of expression in all cell types including skin (Fig. 1) and was therefore chosen as reference mRNA marker in all further qPCR testings.Fig. 1


mRNA-based skin identification for forensic applications.

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

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
© Copyright Policy
Related In: Results  -  Collection

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

Fig1: 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
Mentions: In addition, five commonly used reference genes (ACTB, B2M, GAPDH, PPIB and UBC) [4, 5, 7, 8, 23–26] were tested in skin samples. Of those, the expression of ACTB appeared to be most abundant in skin material (data not shown). When further tested in the various forensically relevant body fluids, ACTB showed similarly high levels of expression in all cell types including skin (Fig. 1) and was therefore chosen as reference mRNA marker in all further qPCR testings.Fig. 1

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