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Successful RNA extraction from various human postmortem tissues

International Journal of Legal Medicine volume 121, pages 136–142 (2007)Cite this article

Abstract

Recently, several authors described the observation that RNA degradation does not correlate with the postmortem interval (PMI), but rather with other parameters like environmental impact and the circumstances of death. Therefore, the question arose if the analysis of gene expression could be a valuable tool in forensic genetics to contribute to the determination of the cause of death. In our study, six human tissues obtained from six individuals with PMI varying between 15 and 118 h were used for total RNA extraction. Quantification was performed using a GAPDH real-time assay, and the quality of mRNA was checked by amplification of different fragment lengths of the GAPDH transcript. In our set of samples, nearly all tissues in all PMI revealed satisfactory results, while skeletal muscle, followed by brain and heart, gave the best results. No correlation between PMI and RNA degradation could be detected, as very good results were observed for all tissues from the individual with the longest PMI. The highly promising results obtained in this study raise hopes that in the near future several fields of forensic investigation may profit from additional information about gene expression patterns and their correlation with pathological findings.

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References

  1. Wilusz CJ, Wormington M, Peltz SW (2001) The cap-to-tail guide to mRNA turnover. Nat Rev Mol Cell Biol 2:237–246

    PubMed  Article  CAS  Google Scholar 

  2. Catts VS, Catts SV, Fernandez HR, Taylor JM, Coulson EJ, Lutze-Mann LH (2005) A microarray study of post-mortem mRNA degradation in mouse brain tissue. Brain Res Mol Brain Res 138:164–177

    PubMed  Article  CAS  Google Scholar 

  3. Preece P, Cairns NJ (2003) Quantifying mRNA in postmortem human brain: influence of gender, age at death, postmortem interval, brain pH, agonal state and inter-lobe mRNA variance. Brain Res Mol Brain Res 118:60–71

    PubMed  Article  CAS  Google Scholar 

  4. Fitzpatrick R, Casey OM, Morris D et al (2002) Postmortem stability of RNA isolated from bovine reproductive tissues. Biochim Biophys Acta 1574:10–14

    PubMed  CAS  Google Scholar 

  5. Kobayashi H, Sakimura K, Kuwano R et al (1990) Stability of messenger RNA in postmortem human brains and construction of human brain cDNA libraries. J Mol Neurosci 2:29–34

    PubMed  CAS  Google Scholar 

  6. Reddy PH, McWeeney S, Park BS et al (2004) Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: upregulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer’s disease. Hum Mol Genet 13:1225–1240

    PubMed  Article  CAS  Google Scholar 

  7. Preece P, Virley DJ, Costandi M et al (2003) An optimistic view for quantifying mRNA in post-mortem human brain. Brain Res Mol Brain Res 116:7–16

    PubMed  Article  CAS  Google Scholar 

  8. Bauer M, Polzin S, Patzelt D (2003) Quantification of RNA degradation by semi-quantitative duplex and competitive RT-PCR: a possible indicator of the age of bloodstains? Forensic Sci Int 138:94–103

    PubMed  Article  CAS  Google Scholar 

  9. Bauer M, Gramlich I, Polzin S, Patzelt D (2003) Quantification of mRNA degradation as possible indicator of postmortem interval—a pilot study. Leg Med (Tokyo) 5:220–227

    CAS  Google Scholar 

  10. Bauer M, Patzelt D (2003) A method for simultaneous RNA and DNA isolation from dried blood and semen stains. Forensic Sci Int 136:76–78

    PubMed  Article  CAS  Google Scholar 

  11. Loddenkötter B, Becker K, Hohoff C, Brinkmann B, Bajanowski T (2005) Real-time quantitative PCR assay for the detection of Helicobacter pylori: no association with sudden infant death syndrome. Int J Legal Med 119:202–206

    PubMed  Article  Google Scholar 

  12. Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39

    PubMed  Article  CAS  Google Scholar 

  13. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    PubMed  Article  CAS  Google Scholar 

  14. Goidin D, Mamessier A, Staquet MJ, Schmitt D, Berthier-Vergnes O (2001) Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal Biochem 295:17–21

    PubMed  Article  CAS  Google Scholar 

  15. Aerts JL, Gonzales MI, Topalian SL (2004) Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR. Biotechniques 36:84–86, 88, 90–91

    PubMed  CAS  Google Scholar 

  16. Phang TW, Shi CY, Chia JN, Ong CN (1994) Amplification of cDNA via RT-PCR using RNA extracted from postmortem tissues. J Forensic Sci 39:1275–1279

    PubMed  CAS  Google Scholar 

  17. Mall G, Eckl M, Sinicina I, Peschel O, Hubig M (2005) Temperature-based death time estimation with only partially known environmental conditions. Int J Legal Med 119:185–194

    PubMed  Article  Google Scholar 

  18. Henssge C, Madea B (2004) Estimation of the time since death in the early post-mortem period. Forensic Sci Int 144:167–175

    PubMed  Article  CAS  Google Scholar 

  19. Takamiya M, Saigusa K, Kumagai R, Nakayashiki N, Aoki Y (2005) Studies on mRNA expression of tissue-type plasminogen activator in bruises for wound age estimation. Int J Legal Med 119:16–21

    PubMed  Article  Google Scholar 

  20. Hayashi T, Ishida Y, Kimura A, Takayasu T, Eisenmenger W, Kondo T (2004) Forensic application of VEGF expression to skin wound age determination. Int J Legal Med 118:320–325

    PubMed  Article  Google Scholar 

  21. Suay L, Salvador ML, Abesha E, Klein U (2005) Specific roles of 5′ RNA secondary structures in stabilizing transcripts in chloroplasts. Nucleic Acids Res 33:4754–4761

    PubMed  Article  CAS  Google Scholar 

  22. Ikematsu K, Tsuda R, Nakasono I (2006) Gene response of mouse skin to pressure injury in the neck region. Leg Med (Tokyo) 8:128–131

    CAS  Google Scholar 

  23. Liu J, Lewohl JM, Dodd PR, Randall PK, Harris RA, Mayfield RD (2004) Gene expression profiling of individual cases reveals consistent transcriptional changes in alcoholic human brain. J Neurochem 90:1050–1058

    PubMed  Article  CAS  Google Scholar 

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Acknowledgement

The authors thank Benedikt Vennemann and Markus Große Perdekamp for help with the sample collection and Manuela Liebers for helpful discussion. This work is part of the bachelor thesis of KM.

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Affiliations

  1. Institute of Legal Medicine, University of Freiburg, Albertstr. 9, 79104, Freiburg, Germany

    Marielle Heinrich, Katja Matt, Sabine Lutz-Bonengel & Ulrike Schmidt

Authors
  1. Marielle Heinrich
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  2. Katja Matt
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  3. Sabine Lutz-Bonengel
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  4. Ulrike Schmidt
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Corresponding author

Correspondence to Marielle Heinrich.

Electronic supplementary material

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Supplementary Table 1

Sequences of the forward primers (in 5′-3′ orientation) for the amplification of different lengths of the GAPDH transcript. For all amplicons, the same reverse primer (5′-tgccctgtagaaattcgttg-3′) was used. The different forward primers were chosen to span at least one exon/exon boundary. E/E Exon/Exon boundary according to GenBank NM002046 (mRNA) and J04038 (genomic sequence) (DOC 29 kb)

Supplementary Fig. 1

a Example of the real-time PCR results of the GAPDH assay using RNA extracted from muscle tissue of the individuals A–E (according to Table 2). b Example of a denaturing agarose gel electrophoresis using RNA extracted from skeletal muscle of individual A. The band representing 28s ribosomal RNA is stronger than the one representing 18s ribosomal RNA indicating a high quality of the extracted RNA (DOC 96 kb)

Supplementary Fig. 2

Example for the amplification of different lengths of the GAPDH transcripts for individual E. a PCR GAPDH_F1–F3. b PCR GAPDH_F4–F5 (DOC 305 kb)

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Heinrich, M., Matt, K., Lutz-Bonengel, S. et al. Successful RNA extraction from various human postmortem tissues. Int J Legal Med 121, 136–142 (2007). https://doi.org/10.1007/s00414-006-0131-9

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  • DOI: https://doi.org/10.1007/s00414-006-0131-9

Keywords

  • mRNA
  • Degradation
  • Quantitative real-time PCR
  • GAPDH
  • Human tissue