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RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays

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Abstract

Messenger RNA (mRNA) profiling in post-mortem human tissue might reveal information about gene expression at the time point of death or close to it. When working with post-mortem human tissue, one is confronted with a natural RNA degradation caused by several parameters which are not yet fully understood. The aims of the present study were to analyse the influence of impaired RNA integrity on the reliability of quantitative gene expression data and to identify ante- and post-mortem parameters that might lead to reduced RNA integrities in post-mortem human brain, cardiac muscle and skeletal muscle tissues. Furthermore, this study determined the impact of several parameters like type of tissue, age at death, gender and body mass index (BMI), as well as duration of agony, cause of death and post-mortem interval on the RNA integrity. The influence of RNA integrity on the reliability of quantitative gene expression data was analysed by generating degradation profiles for three gene transcripts. Based on the deduced cycle of quantification data, this study shows that reverse transcription quantitative polymerase chain reaction (RT-qPCR) performance is affected by impaired RNA integrity. Depending on the transcript and tissue type, a shift in cycle threshold values of up to two cycles was observed. Determining RNA integrity number of 136 post-mortem samples revealed significantly different RNA qualities among the three tissue types with brain revealing significantly lower integrities compared to skeletal and cardiac muscle. The body mass index was found to influence RNA integrity in skeletal muscle tissue (M. iliopsoas). Samples originating from deceased with a BMI > 25 were of significantly lower integrity compared to samples from normal weight donors. Correct data normalisation was found to partly diminish the effects caused by impaired RNA quality. Nevertheless, it can be concluded that in post-mortem tissue with low RNA integrity numbers, the detection of large differences in gene expression activities might still be possible, whereas small expression differences are prone to misinterpretation due to degradation. Thus, when working with post-mortem samples, we recommend generating degradation profiles for all transcripts of interest in order to reveal detection limits of RT-qPCR assays.

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Acknowledgements

The authors would like to extend their special thanks to Elke Troppmann (Institute of Human Genetics, Freiburg University) for the kind permission to use the Nanodrop machine and to Anja Schoepflin (Institute of Pathology, Freiburg University) for the kind permission to use the Bioanalyzer. We are also grateful to Prof. Dr. Dieter Hauschke, Institute of Biometry and Medical Statistics, Freiburg University, for statistical consulting, to Thomas Rost and Wolfgang Schmidt and the autopsy team in Muenster for their help with sample collection, as well as to Dr. Juliane Sanft (Institute of Legal Medicine, Jena University) for helpful comments on the manuscript. This work is part of A.K.’s Ph.D. thesis.

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Authors and Affiliations

  1. Institute of Legal Medicine, Freiburg University Medical Center, Albertstrasse 9, 79104, Freiburg, Germany

    Antje Koppelkamm, Sabine Lutz-Bonengel & Marielle Vennemann

  2. Institute of Forensic Genetics, Im Derdel 8, 48161, Münster, Germany

    Antje Koppelkamm

  3. Centre for Forensic and Legal Medicine, University of Dundee, Dundee, DD1 4HN, Scotland, UK

    Benedikt Vennemann

  4. Institute of Legal Medicine, University of Münster, Röntgenstrasse 23, 48149, Münster, Germany

    Tony Fracasso

  5. University Center of Legal Medicine, University of Geneva, Rue Michel-Servet 1, 1211, Geneva, Switzerland

    Tony Fracasso

  6. Centre for Forensic Science, University of Strathclyde, Royal College, 204 George Street, Glasgow, G1 1XW, Scotland, UK

    Marielle Vennemann

Authors
  1. Antje Koppelkamm
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  2. Benedikt Vennemann
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  3. Sabine Lutz-Bonengel
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  4. Tony Fracasso
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  5. Marielle Vennemann
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Corresponding author

Correspondence to Marielle Vennemann.

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ESM Figure 1

Scatter plots with Pearson correlation (R) between the post-mortem interval (PMI) and RNA integrity numbers (RIN) observed in human post-mortem brain (n = 42), cardiac muscle (n = 43) and skeletal muscle (n = 41) tissue. Analysis was performed for samples with PMI < 50 h. Significances (p) were calculated using SPSS (DOC 110 kb)

ESM Figure 2

Box–whisker plots of RIN values according to the following parameters: a age at death, b gender, c duration of agony and d cause of death (DOC 119 kb)

ESM Table 1

Clinical information including cause of death, agony, gender, age at death, body mass index (BMI), post-mortem interval (PMI), collected tissue type and RIN number of the RNA sample for each of the 49 individuals included in this study (XLS 43 kb)

ESM Table 2

Mean cycles of quantification (C q) with standard deviation (SD) obtained from three assays (ACTB, B2M and 18S rRNA) for artificially degraded, commercially available total RNA from brain (A), heart (B) and skeletal muscle (C). Additionally, the total C q shifts between the highest and the lowest RIN values, respectively, are given. The Pearson correlation (R) significances (p) were calculated using SPSS (DOC 82 kb)

ESM Table 3

Pearson correlation (R) and significances (p) were calculated using SPSS to show correlations between RIN and non-normalised cycles of quantification (NN-C q) and calibrated normalised relative quantities (CNRQ), respectively. Data are given for all ten gene transcripts analysed in Koppelkamm et al. [20] for brain (A), cardiac muscle (B) and skeletal muscle (C) (DOC 56 kb)

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Koppelkamm, A., Vennemann, B., Lutz-Bonengel, S. et al. RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays. Int J Legal Med 125, 573–580 (2011). https://doi.org/10.1007/s00414-011-0578-1

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  • DOI: https://doi.org/10.1007/s00414-011-0578-1

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