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A validation study of mRNA markers for skin cell identification
Forensic Science International: Genetics Supplement Series 4 (2013) e129–e130
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
A validation study of mRNA markers for skin cell identification S.E. Hall a,b,*, R.A.H. van Oorschot a, R.J. Mitchell b, K.N. Ballantyne a a b
Office of the Chief Forensic Scientist, Victoria Police Forensic Services Department, Macleod, Victoria, Australia Department of Genetics, La Trobe University, Melbourne, Victoria, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 September 2013 Accepted 2 October 2013
The objectives of this study were to develop and optimize a multiplex of three skin specific gene markers; loricrin (LOR), corneodesmosin (CDSN) and keratin 9 (KRT9) and 1 house-keeping marker, b-actin (ACTB) using an endpoint PCR assay to analyze expression data from a range of relevant samples. Marker specificity and suitability were evaluated for their inclusion in future forensic casework. The presence of the three skin mRNA markers was successfully confirmed from swabs of human skin obtained from 20 individuals at each of 6 different body sites (forehead, neck, arm, palm, leg and sole). Significant variation was observed in the relative expression of the three genes across the body sites, with some individuals consistently failing to express one or more of the targets. Inter-individual variation was also evident. Accordingly, these markers must be used with caution in the identification of skin in forensic samples. Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic science Body fluid identification Skin mRNA Endpoint PCR
1. Introduction At present, there is a strong trend in forensic genetics for the development of alternative approaches to identifying the cellular origin of biological stains from crime scene samples. Many laboratories are focused on the identification of tissue-specific messenger RNA (mRNA) markers for the development of an assay to detect forensically relevant human body fluids. While skinspecific mRNA transcripts have been described previously [1,2], variation within and between individuals has been observed in some cases, complicating the determination of the presence of skin using these markers, thus pointing to the need for further investigation. The objective of the current work is to develop and optimize a multiplex of three skin specific gene markers; loricrin (LOR), corneodesmosin (CDSN) and keratin 9 (KRT9) and 1 house-keeping marker, b-actin (ACTB) using an endpoint PCR assay for their utilization in future forensic casework. Marker specificity and suitability were evaluated, analysing expression data from a range of relevant samples. 2. Materials and methods Skin samples were obtained from the forehead, neck, arm, palm, leg and sole of 20 individuals. A 6 cm 4 cm DNA/RNA free,
* Corresponding author at: Office of the Chief Forensic Scientist, Victoria Police Forensic Services Department, Macleod, Victoria, Australia. Tel.: +61 431330022. E-mail address: [email protected] (S.E. Hall).
low-adhesive hypo-allergenic template was placed onto each site and skin cells were collected with sterile cotton tipped swabs, moistened with 100 ml RNase-free water. The swabs were rolled within the template 20 times per site with consistent pressure and dried at room temperature. The whole swab from each site was used for RNA extraction. RNA was extracted using Qiagen RNeasy1 Mini kit in a total elution volume of 14 ml, cDNA was synthesized with Superscript1 III First Strand Synthesis System Supermix (Invitrogen), both as per manufacturers’ instructions. The maximum input of RNA extract from skin samples was 8 ml in a 20 ml reaction. A RT negative reaction (containing no reverse transcriptase enzyme) was included. Primers for LOR (F-CTTCCTGGTGCTTTGGGC; R-CCAGAGGTCTTCACGCAGTC) and KRT9 (F-AGCAAGAAAGCAGCTCTGGAGAAGAG; R-GACGTCAGTGATCTGGGCCTCC) were redesigned using online software Primer-BLAST [3] for the purpose of this study because of their poor performance in early trials. Primers were deemed suitable based on their length, melting temperature and selfcomplementarity as well as avoiding the presence of SNPs in forward or reverse sequences and spanning exon-exon junctions to avoid genomic DNA amplification. For CDSN and ACTB the published primers proved satisfactory [1]. Endpoint PCR was performed with custom primers (Invitrogen) and the AmpliTaq Gold1 System (Life Technologies). A multiplex reaction was performed for the amplification of ACTB, CDSN and LOR, whereas KRT9 amplification was performed in singleplex. 2 ml of the RT reaction was amplified in a total reaction volume of 20 ml. All amplifications were performed on a GeneAmp1 9700 PCR System. Product detection and genotyping was analyzed with the 3500xl Genetic Analyzer and GeneMapper ID-X (Life Technologies). 1 ml
1875-1768/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigss.2013.10.066
e130
S.E. Hall et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e129–e130
Table 1 Heat map illustrating expression intensity of the skin-targeted mRNA markers and the housekeeping gene (ACTB) in an endpoint multiplex. The relative expression of each marker is indicated per sample in which each shade of red represents a specific range of expression intensity (++++: >30,000; +++: 10,000–30,000; ++: 1000–10,000; + 175– 1000; *: Below detection threshold [175rfu]; –: not detected).
aliquot of the amplified product was added to 8.9 ml Hi-DiTM formamide and 0.1 ml of GeneScanTM 500 LIZ size standard. 3. Results and discussion The presence of the three skin mRNA markers was successfully confirmed in swabs of human skin obtained from 20 individuals from 6 different body sites (forehead, neck, arm, palm, leg and sole). In some cases the housekeeping gene (ACTB) was not detected (Table 1). These latter samples were excluded from further analysis even when skin markers were detected. Considering the relatively low success rate of ACTB detection, we consider it to be an inappropriate housekeeping gene for evaluating skin cells. CDSN was observed to be the most sensitive marker, with the greatest expression rates detected in swabs from the forehead (95%), neck (73%) and palm (86%). LOR was also detected most frequently in the face and palm, as well as the leg; 61%, 57% and 64% respectively (data not shown). Detection problems were encountered with KRT9, with sporadic detection at low signal intensities in only a few donors. KRT9 was detected on the face, neck and leg of four different individuals. One report [1] suggests that this gene is specifically expressed in the palms and soles of the feet, however we failed to detect KRT9 in any of the donors’ soles (or arms) and only one participant indicated the presence of this marker in their palm. Significant variation within individuals was observed in the relative expression of the three genes across the body sites, with some individuals consistently failing to express
one or more of the targets. Inter-individual variation was also evident. Further work is required to determine if these skintargeted mRNA markers are detectable in other forensically relevant body fluids, as well as assess factors which cause variation in expression between individuals. These markers must be used with caution in the identification of skin cells in forensic samples.
Conflict of interest None. Acknowledgements Thanks to individuals from VPFSD who donated their time and samples to this study. Special acknowledgment to Adam Poy for technical assistance with 3500xl Genetic Analyzer and GeneMapper IDX.
References [1] M. Visser, D. Zubakov, K.N. Ballantyne, et al., mRNA-based skin identification for forensic applications, Int. J. Legal Med. 125 (2011) 253–263. [2] A. Lindenbergh, M. de Pagter, G. Ramdayal, et al., A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact trace, Forensic Sci. Int. Genet. 6 (2012) 565–577. [3] J. Ye, G. Coulouris, I. Zaretskaya, et al., Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction, BMC Bioinformatics 13 (2012) 134.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
A validation study of mRNA markers for skin cell identification S.E. Hall a,b,*, R.A.H. van Oorschot a, R.J. Mitchell b, K.N. Ballantyne a a b
Office of the Chief Forensic Scientist, Victoria Police Forensic Services Department, Macleod, Victoria, Australia Department of Genetics, La Trobe University, Melbourne, Victoria, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 September 2013 Accepted 2 October 2013
The objectives of this study were to develop and optimize a multiplex of three skin specific gene markers; loricrin (LOR), corneodesmosin (CDSN) and keratin 9 (KRT9) and 1 house-keeping marker, b-actin (ACTB) using an endpoint PCR assay to analyze expression data from a range of relevant samples. Marker specificity and suitability were evaluated for their inclusion in future forensic casework. The presence of the three skin mRNA markers was successfully confirmed from swabs of human skin obtained from 20 individuals at each of 6 different body sites (forehead, neck, arm, palm, leg and sole). Significant variation was observed in the relative expression of the three genes across the body sites, with some individuals consistently failing to express one or more of the targets. Inter-individual variation was also evident. Accordingly, these markers must be used with caution in the identification of skin in forensic samples. Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic science Body fluid identification Skin mRNA Endpoint PCR
1. Introduction At present, there is a strong trend in forensic genetics for the development of alternative approaches to identifying the cellular origin of biological stains from crime scene samples. Many laboratories are focused on the identification of tissue-specific messenger RNA (mRNA) markers for the development of an assay to detect forensically relevant human body fluids. While skinspecific mRNA transcripts have been described previously [1,2], variation within and between individuals has been observed in some cases, complicating the determination of the presence of skin using these markers, thus pointing to the need for further investigation. The objective of the current work is to develop and optimize a multiplex of three skin specific gene markers; loricrin (LOR), corneodesmosin (CDSN) and keratin 9 (KRT9) and 1 house-keeping marker, b-actin (ACTB) using an endpoint PCR assay for their utilization in future forensic casework. Marker specificity and suitability were evaluated, analysing expression data from a range of relevant samples. 2. Materials and methods Skin samples were obtained from the forehead, neck, arm, palm, leg and sole of 20 individuals. A 6 cm 4 cm DNA/RNA free,
* Corresponding author at: Office of the Chief Forensic Scientist, Victoria Police Forensic Services Department, Macleod, Victoria, Australia. Tel.: +61 431330022. E-mail address: [email protected] (S.E. Hall).
low-adhesive hypo-allergenic template was placed onto each site and skin cells were collected with sterile cotton tipped swabs, moistened with 100 ml RNase-free water. The swabs were rolled within the template 20 times per site with consistent pressure and dried at room temperature. The whole swab from each site was used for RNA extraction. RNA was extracted using Qiagen RNeasy1 Mini kit in a total elution volume of 14 ml, cDNA was synthesized with Superscript1 III First Strand Synthesis System Supermix (Invitrogen), both as per manufacturers’ instructions. The maximum input of RNA extract from skin samples was 8 ml in a 20 ml reaction. A RT negative reaction (containing no reverse transcriptase enzyme) was included. Primers for LOR (F-CTTCCTGGTGCTTTGGGC; R-CCAGAGGTCTTCACGCAGTC) and KRT9 (F-AGCAAGAAAGCAGCTCTGGAGAAGAG; R-GACGTCAGTGATCTGGGCCTCC) were redesigned using online software Primer-BLAST [3] for the purpose of this study because of their poor performance in early trials. Primers were deemed suitable based on their length, melting temperature and selfcomplementarity as well as avoiding the presence of SNPs in forward or reverse sequences and spanning exon-exon junctions to avoid genomic DNA amplification. For CDSN and ACTB the published primers proved satisfactory [1]. Endpoint PCR was performed with custom primers (Invitrogen) and the AmpliTaq Gold1 System (Life Technologies). A multiplex reaction was performed for the amplification of ACTB, CDSN and LOR, whereas KRT9 amplification was performed in singleplex. 2 ml of the RT reaction was amplified in a total reaction volume of 20 ml. All amplifications were performed on a GeneAmp1 9700 PCR System. Product detection and genotyping was analyzed with the 3500xl Genetic Analyzer and GeneMapper ID-X (Life Technologies). 1 ml
1875-1768/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigss.2013.10.066
e130
S.E. Hall et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e129–e130
Table 1 Heat map illustrating expression intensity of the skin-targeted mRNA markers and the housekeeping gene (ACTB) in an endpoint multiplex. The relative expression of each marker is indicated per sample in which each shade of red represents a specific range of expression intensity (++++: >30,000; +++: 10,000–30,000; ++: 1000–10,000; + 175– 1000; *: Below detection threshold [175rfu]; –: not detected).
aliquot of the amplified product was added to 8.9 ml Hi-DiTM formamide and 0.1 ml of GeneScanTM 500 LIZ size standard. 3. Results and discussion The presence of the three skin mRNA markers was successfully confirmed in swabs of human skin obtained from 20 individuals from 6 different body sites (forehead, neck, arm, palm, leg and sole). In some cases the housekeeping gene (ACTB) was not detected (Table 1). These latter samples were excluded from further analysis even when skin markers were detected. Considering the relatively low success rate of ACTB detection, we consider it to be an inappropriate housekeeping gene for evaluating skin cells. CDSN was observed to be the most sensitive marker, with the greatest expression rates detected in swabs from the forehead (95%), neck (73%) and palm (86%). LOR was also detected most frequently in the face and palm, as well as the leg; 61%, 57% and 64% respectively (data not shown). Detection problems were encountered with KRT9, with sporadic detection at low signal intensities in only a few donors. KRT9 was detected on the face, neck and leg of four different individuals. One report [1] suggests that this gene is specifically expressed in the palms and soles of the feet, however we failed to detect KRT9 in any of the donors’ soles (or arms) and only one participant indicated the presence of this marker in their palm. Significant variation within individuals was observed in the relative expression of the three genes across the body sites, with some individuals consistently failing to express
one or more of the targets. Inter-individual variation was also evident. Further work is required to determine if these skintargeted mRNA markers are detectable in other forensically relevant body fluids, as well as assess factors which cause variation in expression between individuals. These markers must be used with caution in the identification of skin cells in forensic samples.
Conflict of interest None. Acknowledgements Thanks to individuals from VPFSD who donated their time and samples to this study. Special acknowledgment to Adam Poy for technical assistance with 3500xl Genetic Analyzer and GeneMapper IDX.
References [1] M. Visser, D. Zubakov, K.N. Ballantyne, et al., mRNA-based skin identification for forensic applications, Int. J. Legal Med. 125 (2011) 253–263. [2] A. Lindenbergh, M. de Pagter, G. Ramdayal, et al., A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact trace, Forensic Sci. Int. Genet. 6 (2012) 565–577. [3] J. Ye, G. Coulouris, I. Zaretskaya, et al., Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction, BMC Bioinformatics 13 (2012) 134.