A time course study demonstrating mRNA, microRNA, 18S rRNA, and U6 snRNA changes to estimate PMI in deceased rat's spleen
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Ye-hui Lv1, Kai-jun Ma, Heng Zhang
1Department of Forensic Medicine, Shanghai Medical School of Fudan University, 200032, Shanghai, China.
Abstract:
Determining the postmortem interval (PMI) is important in criminal, civil, and forensic cases. We examined the feasibility of using the transcript abundances of mRNAs, 18S rRNA, U6 snRNA, and microRNAs as a means to estimate the PMI. We removed spleen tissues from rats at different PMIs under 4°C or 25°C and examined gene transcript abundances in these samples by RT-qPCR. Using the algorithm geNorm, we found that microRNAs to be appropriate control markers because they were less affected by PMI and temperature. We also characterized relationships between observed PMI and the transcript levels of the above-mentioned RNAs. GAPDH1 and ACTB1 fluctuated slightly like cubic curves, while GAPDH2 and ACTB2 decreased rapidly. 18S rRNA transcript level exhibited a parabolic-like trend at 25°C and exponential growth at 4°C, while U6 transcript level exhibited exponential decay at 25°C and a parabolic-like trend at 4°C. Following validation, we conclude that GAPDH2, ACTB2, and 18S rRNA are suitable makers in the accurate determination of PMI.
Insights
Accurate postmortem interval (PMI) estimation is crucial. This study identifies specific RNA markers, including GAPDH2, ACTB2, and 18S rRNA, for precise PMI determination in forensic science.
Area of Science:
- Forensic Science
- Molecular Biology
- Biochemistry
Background:
- Accurate postmortem interval (PMI) determination is vital in legal and forensic investigations.
- RNA transcript abundance is a potential biomarker for estimating PMI, but its stability varies.
- Understanding RNA stability under different postmortem conditions is essential for reliable PMI estimation.
Purpose of the Study:
- To evaluate the feasibility of using various RNA transcript abundances (mRNAs, 18S rRNA, U6 snRNA, microRNAs) for estimating PMI.
- To identify reliable RNA markers for accurate PMI determination in forensic casework.
- To assess the influence of temperature on RNA transcript stability and PMI estimation.
Main Methods:
- Spleen tissues from rats were collected at various postmortem intervals and stored at 4°C or 25°C.
- Gene transcript abundances were quantified using quantitative reverse transcription PCR (RT-qPCR).
- The geNorm algorithm was employed to identify stable reference RNA markers, and transcript level trends were analyzed against PMI and temperature.
Main Results:
- MicroRNAs were identified as stable control markers, minimally affected by PMI and temperature variations.
- GAPDH1 and ACTB1 mRNA levels showed minor cubic fluctuations, while GAPDH2 and ACTB2 mRNA levels decreased rapidly.
- 18S rRNA and U6 snRNA transcript levels exhibited distinct trends (parabolic, exponential decay/growth) influenced by temperature and PMI.
Conclusions:
- GAPDH2, ACTB2, and 18S rRNA were validated as suitable and accurate markers for PMI determination.
- MicroRNAs serve as reliable reference controls for normalizing RNA expression data in PMI studies.
- This research provides a molecular toolkit for improving the accuracy of postmortem interval estimation in forensic investigations.
