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Evaluating the potential of housekeeping genes, rRNAs, snRNAs, microRNAs and circRNAs as reference genes for the estimation of PMI

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Abstract

The precise estimation of postmortem interval (PMI) is a critical step in death investigation of forensic cases. Detecting the degradation of RNA in tissues by real time quantitative polymerase chain reaction (RT-qPCR) technology provides a new theoretical basis for estimation of PMI. However, most commonly used reference genes degrade over time, while previous studies seldom consider this when selecting suitable reference genes for the estimation of PMI. Studies have shown microRNAs (miRNAs) are very stable and circular RNAs (circRNAs) have recently emerged as a novel class of RNAs with high stability. We aimed to evaluate the stability of the two kinds of RNAs and normal reference genes using geNorm and NormFinder algorithms to identify tissue-specific reference genes for PMI estimation. The content of candidate RNAs from mouse heart, liver and skeletal muscle tissues were dynamically examined in 8 consecutive days after death. Among the 11 candidate genes (β-actin, Gapdh, Rps18, 5S, 18S, U6, miR-133a, miR-122, circ-AFF1, LC-Ogdh and LC-LRP6), the following genes showed prioritized stability: miR-122, miR-133a and 18S in heart tissues; LC-Ogdh, circ-AFF1 and miR-122 in liver tissues; and miR-133a, circ-AFF1 and LC-LRP6 in skeletal muscle tissues. Our results suggested that miRNAs and circRNAs were more stable as reference genes than other kinds of RNAs regarding PMI estimation. The appropriate internal control genes were not completely the same across tissue types.

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Funding

This work was financially sponsored by the Opening Foundation of Shanghai Key Laboratory of Crime Scene Evidence (grant No.XCWZ20).

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Authors and Affiliations

  1. Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, 138 Yixueyuan Road, Xuhui District, Shanghai, 200032, People’s Republic of China

    Chunyan Tu, Tieshuai Du, Chengchen Shao, Zengjia Liu, Liliang Li & Yiwen Shen

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  1. Chunyan Tu
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  2. Tieshuai Du
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  3. Chengchen Shao
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  4. Zengjia Liu
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  5. Liliang Li
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Correspondence to Liliang Li or Yiwen Shen.

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Conflict of interest

All authors declare that they have no conflict ofinterest. The authors want to thank Professor Jian-Hui Xie from the Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University for his assistance in screening potential circRNAs.

Ethical approval

All applicable international, national, and/or institutionalguidelines for the care and use of animals were followed. Allprocedures performed in studies involving animals were in accordance with the Ethical Review Board at the School of Basic Medical Sciences, Fudan University.

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Tu, C., Du, T., Shao, C. et al. Evaluating the potential of housekeeping genes, rRNAs, snRNAs, microRNAs and circRNAs as reference genes for the estimation of PMI. Forensic Sci Med Pathol 14, 194–201 (2018). https://doi.org/10.1007/s12024-018-9973-y

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  • DOI: https://doi.org/10.1007/s12024-018-9973-y

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