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Degradation of AIF in mouse heart tissue for estimating postmortem interval (PMI)

  • Duo Peng
    Affiliations
    Department of Forensic Genetics, West School of Basic Science and Forensic Medicine, Sichuan University, Chengdu, 610081, Sichuan, China
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  • Zhilong Li
    Affiliations
    Department of Forensic Genetics, West School of Basic Science and Forensic Medicine, Sichuan University, Chengdu, 610081, Sichuan, China
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  • Huan Tian
    Affiliations
    Department of Forensic Genetics, West School of Basic Science and Forensic Medicine, Sichuan University, Chengdu, 610081, Sichuan, China
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  • Dan Chen
    Affiliations
    Department of Forensic Genetics, Institute of Forensic Science, Chengdu Public Security Bureau, Chengdu, 610081, Sichuan, China
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  • Jijun Wang
    Affiliations
    DNA Lab of HI-TECH Industrial Sub-branch of Chengdu Municipal Public Security Bureau, Chengdu, 610081, Sichuan, China
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  • Peng Bai
    Affiliations
    Department of Forensic Genetics, West School of Basic Science and Forensic Medicine, Sichuan University, Chengdu, 610081, Sichuan, China
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  • Weibo Liang
    Correspondence
    Corresponding authors at: Institute for Forensic Medicine, Sichuan University, Chengdu, Sichuan, China.
    Affiliations
    Department of Forensic Genetics, West School of Basic Science and Forensic Medicine, Sichuan University, Chengdu, 610081, Sichuan, China
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  • Lin Zhang
    Correspondence
    Corresponding authors at: Institute for Forensic Medicine, Sichuan University, Chengdu, Sichuan, China.
    Affiliations
    Department of Forensic Genetics, West School of Basic Science and Forensic Medicine, Sichuan University, Chengdu, 610081, Sichuan, China
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Published:September 29, 2017DOI:https://doi.org/10.1016/j.fsigss.2017.09.224

      Abstract

      Estimating time of death in forensic routines is particularly important, and a range of time can be determined by traditional methods Degradation of mRNA has been used to estimate postmortem interval (PMI) and many potential mRNA markers are found in previous studies. In this study, we aimed to find a new mRNA marker which can determine PMI with its degradation. Besides, our previous results proposed that DNA was suitable as normalization. So, in this study we determined apoptosis inducing factor (AIF) degradation against Caspase-3 DNA to see if AIF is an effective marker to estimate time of death. To do that, corpses of mice in 37 °C were simulating the environment of summer days in Chengdu and heart tissue was obtained in different time points. RNA and DNA were co-extracted with Bioteke co-extraction kits followed by expression level detection with RT-qPCR of MiScript SYBR Green PCR Kit. The results showed that AIF was slowly degraded in 48 h PMI. What’s more, AIF represented a faster degradation process and a good liner correlation within 14–20 h PMI. Therefore, we suggested AIF was an useful marker to estimate time of death in14–20 h PMI.

      Keywords

      1. Introduction

      Estimating time of death is particularly important in forensic cases because it can provide effective clues for crime investigation, and contribute to rebuilding case scene. Traditional ways of estimating postmortem interval (PMI) such as corpse phenomena, forensic entomology and tissue/body fluids changes only obtain a rang time of death [
      • Bauer M.
      • Gramlich I.
      • Polzin S.
      • Patzelt D.
      Quantification of mRNA degradation as possible indicator of postmortem interval—a pilot study.
      ,
      • Sampaio-Silva F.
      • ̃es T.M.
      • Carvalho F.l.
      • Dinis-Oliveira R.J.
      • Silvestre R.
      Profiling of RNA degradation for estimation of post morterm interval.
      ]. To obtain relative accurate time of death, molecular (like DNA and RNA) methods has been used to estimate PMI. Degradation of messenger RNA (mRNA) after death was considered as a promising method to estimate time of death and several mRNA markers were found to be useful to estimate PMI in previous studies [
      • Lv Y.H.
      • Ma J.L.
      • Pan H.
      • Zhang H.
      • He M.
      • Zhang P.
      • et al.
      A time course study demonstrating mRNA, microRNA, 18S rRNA, and U6 snRNA changes to estimate PMI in deceased rat’s spleen.
      ]. Apoptosis inducing factor (AIF) is reported to mediate cell death in some biological mechanisms [
      • Candé C.
      • Cohen I.
      • Daugas E.
      • Luigi Ravagnan N.L.
      • Zamzami N.
      • Kroemer G.
      Apoptosis-inducing factor (AIF)a novel caspase independent death.
      ,
      • Sun H.
      • Yang S.
      • Li J.
      • Zhang Y.
      • Gao D.
      • Zhao S.
      Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death.
      ] so we speculate that AIF might be a promising marker to estimate 48 h PMI. Besides, previous studies demonstrated that DNA can be as normalized gene in mRNA degradation [
      • Deng W.
      • Lv M.
      • Wang L.
      • Bai P.
      • Liang W.
      • Zhang L.
      mRNA degradation pattern analysis in post-mortem normalized using the DNA.
      ]. Therefore, in this study we determined mRNA of AIF degradation against DNA of Caspase-3 in mice heart tissue in 48 h PMI, and established a statistical model to see whether the mRNA of AIF is an effective marker to estimate time of death.

      2. Material and methods

      A total of 87 healthy male mice were obtained from Animal Experimental Center of Basic Science and Forensic Medicine in SiChuan University after obtaining ethical approval from the Applied Science Ethics Panel. And then mice were euthanized by cervical dislocation and placed in 37 °C incubator to simulate environment of summer days in Chengdu. After that we set 29 time points of PMI, including 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 36, 48 h PMI. Altogether, there were 87 heart samples in our study and stored in −80 °C.
      DNA and RNA were co-extracted from samples with commercial kit and followed by reverse transcription. The cDNA was quantified with SYBR Green relative quantification on ABI 7500 real-time PCR and Cq values were obtained with ABI 7500 software. Primer of AIF (mRNA) and Caspase-3 (DNA) were designed by Primer 5 software and verified with UCSC In-Silico PCR.

      3. Results and discussion

      Each sample was replicated twice in qPCR and dCq values were acquired as dCq = CqmRNA – CqDNA. The relationship of dCq values and 48 h PMI was shown in Fig. 1. As Fig. 1 showed, dCq values increased within 48 h PMI. This indicated that AIF was degraded after death in 48 h PMI. Based on the correlation of dCq and PMI, we set up a statistical model: y=0.291x+2.558; R2=0.653 (Fig. 1). The slope of the statistical model formula suggested that AIF degraded slowly in whole 48 h PMI. AIF is a factor which induced apoptosis and there would be an increased transcription in time of cell death. So, we assumed that lower slope in 48 h PMI was caused by the balance of its functional synthesis and degradation.
      Fig. 1
      Fig. 1Correlation of AIF dCq and 48 h PMI in heart. All error bars represented standard deviation. Black liner was trend line and the statistical model we simulated was y=0.291x+2.558; R2=0.653.
      However, a better liner relationship (y=1.822x+4.114; R2=0.969) was presented in 14–20 h PMI, showed in Fig. 2. The slop of this formula indicated AIF was faster degraded within 14–20 h PMI. This maybe because the rate of degradation was faster than synthesis in this period. R2 was much higher in Fig. 2, suggesting that AIF was an effective marker for 14–20 h PMI determination.
      Fig. 2
      Fig. 2Correlation of AIF dCq and 14–20 h PMI in heart. All error bars represented standard deviation. Black liner was trend line and the statistical model we simulated was y=1.822x+4.114; R2=0.969.

      4. Conclusions

      In this study, we acquired correlation of AIF degradation against Caspase-3 with 48 h PMI and then established a statistical model. The results in our study suggested that AIF degradation was helpful to estimate 14–20 h PMI.

      5. Conflict of interest

      None.

      6. Role of funding

      Grants ( No.81471827 ) from the National Natural Science Foundation of China .

      7 Acknowledgement

      None.

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