Collaborative EDNAP exercises on messenger RNA/DNA co-analysis for body fluid identification (blood, saliva, semen) and STR profiling

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
• 3. Results and discussion
• Role of funding
• Conflict of interest
• Acknowledgements
• References
• Copyright

Abstract 

RNA profiling is a new method for the identification of forensically relevant biological stains, such as blood, saliva, semen, vaginal secretions, menstrual blood, sweat and skin. To demonstrate the suitability of mRNA profiling for use in forensic casework, three collaborative exercises on RNA analysis or RNA/DNA co-analysis for body fluid identification (blood, saliva, semen) and STR profiling were organized within the European DNA Profiling Group (EDNAP). The results of these collaborative exercises support the potential use of an mRNA-based system for the identification of body fluids along with conventional DNA profiling.

Keywords: Forensic science, mRNA Profiling, Body fluid identification, Blood, Saliva, Semen

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1. Introduction 

Conventional methods for body fluid stain analysis involve the use of enzymatic or immunologic tests. Due to limited specificity (i.e. cross-reactivity with other species or tissues), many of these tests are presumptive in nature. mRNA profiling is a new method for the identification of body fluids and is considered a confirmatory test that conclusively identifies a body fluid. A number of mRNA markers have been identified for the forensically most relevant body fluids: blood, saliva, semen, vaginal secretions and menstrual blood [1]. Three collaborative exercises on mRNA profiling for the identification of blood, saliva and semen were organized by EDNAP to implement the mRNA profiling method in the participating laboratories and to evaluate the most suitable mRNA markers [2], [3]. The organizing laboratories (Institute of Legal Medicine, Zürich and National Center for Forensic Science, Orlando) pre-evaluated published and unpublished mRNA markers for specificity and sensitivity and selected the best candidates (Table 1). Singleplex and multiplex conditions were optimized.

Table 1. List of evaluated mRNA markers for the identification of blood, saliva and semen.

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2. Materials and methods 

Each study included an evaluation of mock casework samples and body fluid dilution series. Participating laboratories were also asked to examine a selected few of bona fide or mock casework samples. Primer mixes, stains and instructions were provided by the organizing laboratories. Total RNA and DNA were co-extracted from the same stain, with an extraction kit or manual method of choice. The RNA extract received DNase treatment to remove residual DNA. RNA quantitation was optional. mRNA was reverse transcribed (RT) into complementary DNA (cDNA), using random primers and an RT kit of choice. cDNA was then amplified with fluorescence-labelled tissue-specific primers and amplicons were separated and detected with capillary electrophoresis (CE).

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3. Results and discussion 

A majority of the 16–18 participating laboratories were able to confirm the presence of the respective body fluid by mRNA profiling and to obtain autosomal STR profiles of the stain donors. Despite some expected variation in sensitivity between laboratories, the reproducibility and sensitivity of the mRNA profiling method was demonstrated using a range of analysis strategies.

Significantly, the blood, saliva and semen markers showed a high degree of specificity with no relevant cross-reaction with other human tissues/body fluids or with other species. The corresponding mRNA markers could be detected in samples as small as 0.001μl blood, 0.05μl saliva and 0.01μl semen (Table 2). Also, challenging mock casework and casework samples were analyzed successfully.

Table 2. Results of the body fluid dilution series, providing an indication of the sensitivity of the mRNA markers. Grey squares: more than half of the laboratories detected this marker; white squares: less than half of the laboratories detected this marker.

The quantity and quality of co-extracted DNA was also sufficient for casework and environmentally exposed samples, even though the yield might be lower compared to non co-extraction methods.

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Role of funding 

No funding.

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Conflict of interest 

None.

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Acknowledgements 

The authors thank the participants for their contribution: M.J. Anjos, R. Banemann, A.M. Bento, A. Berti, E. Borges, C. Bouakaze, A. Carracedo, M. Carvalho, V. Castella, A. Choma, C. Courts, G. De Cock, M. Dötsch, M. Durianciková, B. Hjort, P. Hoff-Olsen, C. Hohoff, P. Johansen, F. Kohlmeier, P.A. Lindenbergh, B. Loddenkötter, B. Ludes, O. Maroñas, D. Moore, M.-L. Morerod, H. Niederstätter, F. Noel, W. Parson, G. Patel, C. Popielarz, A. Roeder, Y. Ruiz, E. Salata, P.M. Schneider, T. Sijen, B. Sviezená, M. Turanská, M. Vennemann, L. Zatkalíková.

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References 

1. Juusola J, Ballantyne J. Multiplex mRNA profiling for the identification of body fluids. Forensic Sci. Int. 2005;152:1–12
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2. Haas C, Hanson E, et al. mRNA profiling for the identification of blood - Results of a collaborative EDNAP exercise. Forensic Sci. Int. Genetics. 2011;5:21–26
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3. C. Haas, E. Hanson, et al., RNA/DNA co-analysis from blood stains—results of a second collaborative EDNAP exercise, Forensic Sci. Int. Genetics, doi:10.1016/j.fsigen.2011.02.004.
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