Advertisement

International Journal of Legal Medicine

, Volume 127, Issue 5, pp 891–900 | Cite as

Development of a mRNA profiling multiplex for the inference of organ tissues

  • Alexander Lindenbergh
  • Margreet van den Berge
  • Roelof-Jan Oostra
  • Cindy Cleypool
  • Annette Bruggink
  • Ate Kloosterman
  • Titia Sijen
Original Article

Abstract

Forensic characterisation of organ tissue generally occurs through histological and immunological assays of limited sensitivity. Here, we explore an alternative approach and examine a total of 41 candidate mRNA markers for their ability to differentiate between brain, lung, liver, skeletal muscle, heart, kidney and skin. Various selection rounds are applied involving 85 organ tissues (36 excised autopsy specimens and 49 frozen tissue sections, with at least ten specimens for each organ type), 20 commercially available RNAs from different human tissues and at least two specimens of blood, saliva, semen, vaginal mucosa, menstrual secretion or touch samples. Finally, 14 markers are regarded tissue-specific and included in an endpoint RT-PCR multiplex together with one general muscle, one blood and one housekeeping marker. This 17-plex is successfully used to analyse a blind test set of 20 specimens including mixtures, and samples derived from stabbing of organ tissues. With the blind test set samples, it is shown that an earlier described interpretation strategy for RNA cell typing results [1] is also effective for tissue inference. As organ-typing is embedded in a procedure of combined DNA/RNA extraction and analysis, both donor and organ type information is derived from the same sample. Some autopsy specimens presented DNA profiles characteristic for degraded DNA. Nevertheless, the organ-typing multiplex could generate full RNA profiles, which is probably due to small sizes of the amplicons. This assay provides a novel tool for analysis of samples from violent crimes.

Keywords

Forensic science RNA profiling Organ typing Tissue identification Autopsy specimens 

Notes

Acknowledgements

The authors are very grateful to all donors from whom tissues have been used in this study. We thank Kees Krijgsman and Eppo van Houten for excising the autopsy samples. Role of funding: this study was supported by a grant from the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO) within the framework of the Forensic Genomics Consortium Netherlands (FGCN). The work leading to these results also received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 285487 (EUROFORGEN-NoE). We thank Lindy Clarisse for technical assistance and Antoinette Westen for assistance with the manuscript.

Supplementary material

414_2013_895_MOESM1_ESM.doc (376 kb)
ESM 1 (DOC 375 kb)

References

  1. 1.
    Lindenbergh A, Maaskant P, Sijen T (2013) Implementation of RNA profiling in forensic casework. Forensic Sci Int Genet 7:159–166PubMedCrossRefGoogle Scholar
  2. 2.
    Karger B, Meyer E, DuChesne A (1997) STR analysis on perforating FMJ bullets and a new VWA variant allele. Int J Legal Med 110:101–103PubMedCrossRefGoogle Scholar
  3. 3.
    Dieltjes P, Mieremet R, Zuniga S, Kraaijenbrink T, Pijpe J, de Knijff P (2011) A sensitive method to extract DNA from biological traces present on ammunition for the purpose of genetic profiling. Int J Legal Med 125:597–602PubMedCrossRefGoogle Scholar
  4. 4.
    Visser R (1998) Bullet cytology; results of a pilot study. Sixth Cross Channel Conference on Forensic Medicine p11Google Scholar
  5. 5.
    Takata T, Miyaishi S, Kitao T, Ishizu H (2004) Identification of human brain from a tissue fragment by detection of neurofilament proteins. Forensic Sci Int 144:1–6PubMedCrossRefGoogle Scholar
  6. 6.
    Kimura A, Ikeda H, Yasuda S, Yamaguchi K, Tsuji T (1995) Brain tissue identification based on myosin heavy chain isoforms. Int J Legal Med 107:193–196PubMedCrossRefGoogle Scholar
  7. 7.
    Seo Y, Kakizaki E, Takahama K (1997) A sandwich enzyme immunoassay for brain S-100 protein and its forensic application. Forensic Sci Int 87:145–154PubMedCrossRefGoogle Scholar
  8. 8.
    Takahama K (1996) Forensic application of organ-specific antigens. Forensic Sci Int 80:63–69PubMedCrossRefGoogle Scholar
  9. 9.
    Nichols CA, Sens MA (1990) Recovery and evaluation by cytologic techniques of trace material retained on bullets. Am J Forensic Med Pathol 11:17–34PubMedCrossRefGoogle Scholar
  10. 10.
    Fleming RI, Harbison S (2010) The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids. Forensic Sci Int Genet 4:244–256PubMedCrossRefGoogle Scholar
  11. 11.
    Haas C, Klesser B, Maake C, Bar W, Kratzer A (2009) mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Sci Int Genet 3:80–88PubMedCrossRefGoogle Scholar
  12. 12.
    Hanson E, Haas C, Jucker J, Ballantyne J (2011) Identification of skin in touch/contact forensic samples by messenger RNA profiling. Forensic Sci Int Genetics Supplement Series 3:305–306CrossRefGoogle Scholar
  13. 13.
    Hanson E, Haas C, Jucker R, Ballantyne J (2012) Specific and sensitive mRNA biomarkers for the identification of skin in ‘touch DNA’ evidence. Forensic Sci Int Genet 6:548–558PubMedCrossRefGoogle Scholar
  14. 14.
    Hanson EK, Lubenow H, Ballantyne J (2009) Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Anal Biochem 387:303–314PubMedCrossRefGoogle Scholar
  15. 15.
    Juusola J, Ballantyne J (2007) mRNA profiling for body fluid identification by multiplex quantitative RT-PCR. J Forensic Sci 52:1252–1262PubMedGoogle Scholar
  16. 16.
    Lindenbergh A, de Pagter M, Ramdayal G, Visser M, Zubakov D, Kayser M, Sijen T (2012) A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces. Forensic Sci Int Genet 6:565–577PubMedCrossRefGoogle Scholar
  17. 17.
    Zubakov D, Hanekamp E, Kokshoorn M, van IJcken W, Kayser M (2008) Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples. Int J Legal Med 122:135–142PubMedCrossRefGoogle Scholar
  18. 18.
    Zubakov D, Kokshoorn M, Kloosterman A, Kayser M (2009) New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains. Int J Legal Med 123:71–74PubMedCrossRefGoogle Scholar
  19. 19.
    Lee HY, Park MJ, Choi A, An JH, Yang WI, Shin KJ (2011) Potential forensic application of DNA methylation profiling to body fluid identification. Int J Legal Med 126:55–62PubMedCrossRefGoogle Scholar
  20. 20.
    Inoue H, Kimura A, Tuji T (2002) Degradation profile of mRNA in a dead rat body: basic semi-quantification study. Forensic Sci Int 130:127–132PubMedCrossRefGoogle Scholar
  21. 21.
    Wu C, Orozco C, Boyer J, Leglise M, Goodale J, Batalov S, Hodge CL, Haase J, Janes J, Huss JW III, Su AI (2009) BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol 10:R130PubMedCrossRefGoogle Scholar
  22. 22.
    Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinforma 13:134CrossRefGoogle Scholar
  23. 23.
    Flicek P et al (2012) Ensembl 2012. Nucl Acid Res 40:84–90CrossRefGoogle Scholar
  24. 24.
    Nicklas JA, Buel E (2006) Simultaneous determination of total human and male DNA using a duplex real-time PCR assay. J Forensic Sci 51:1005–1015PubMedCrossRefGoogle Scholar
  25. 25.
    Visser M, Zubakov D, Ballantyne KN, Kayser M (2011) mRNA-based skin identification for forensic applications. Int J Legal Med 125:253–263PubMedCrossRefGoogle Scholar
  26. 26.
    Harteveld J, Lindenbergh A, Sijen T (2013) RNA cell typing and DNA profiling of mixed samples: Can cell types and donors be associated? Sci. Justice, http://dx.doi.org/ 10.1016/j.scijus.2013.02.001

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Alexander Lindenbergh
    • 1
  • Margreet van den Berge
    • 1
  • Roelof-Jan Oostra
    • 2
  • Cindy Cleypool
    • 3
  • Annette Bruggink
    • 4
  • Ate Kloosterman
    • 1
  • Titia Sijen
    • 1
  1. 1.Department of Human Biological TracesNetherlands Forensic InstituteThe HagueThe Netherlands
  2. 2.Department of Anatomy Embryology and Physiology, Academic Medical CenterUniversity of AmsterdamAmsterdamThe Netherlands
  3. 3.Department of AnatomyUniversity Medical Center UtrechtUtrechtThe Netherlands
  4. 4.UMC Utrecht BiobankUniversity Medical Center UtrechtUtrechtThe Netherlands

Personalised recommendations