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Estimation of Early Postmortem Interval with β-actin mRNA Degradation in Rat’s Brain, Heart and Kidney

Author: LiuYueLin
Tutor: ZhaoZiQin
School: Fudan University
Course: Forensic
Keywords: Forensic Pathology Early death Real-time quantitative RT-PCR β-actin mRNA
CLC: D919
Type: Master's thesis
Year: 2011
Downloads: 44
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Abstract

Dead time (time of death) has been inferred forensic pathology research hotspot and difficult. Time of death, also known as the elapsed time after the death of (Postmortem Interval, PMI), which found that the check occurs when the body away from the death of the time interval. Based on changes in the body after death has occurred and the demand for forensic practice, the time of death is usually divided into early and late time of death death. Both the limit of death about 24h. Obviously, with respect to the late time of death, the accuracy of early postmortem interval case for practical examination is even more significant. However, the current can be applied in practice in the early postmortem interval method is still only the use of the phenomenon of early corpse (dead cold, Shiban, rigor mortis, bounce reactions) and degree of digestion of stomach contents and other relatively rough empirical, subjective methods . In laboratory studies, the postmortem interval studies involving multiple disciplines and areas, such as the determination of blood, vitreous humor ion concentration of the chemical method to observe changes in tissue morphology immunohistochemistry to study the growth and development of plants and animals forensic insect science and forensic botany methods, the use of CT, MRI, MRS imaging methods, and by studying nucleic acids, proteins of the time-dependent degradation of molecular biology methods. Wherein, in the field of molecular biology, in particular nucleic acid of mRNA using the time-dependent degradation of inference time of death in recent years research hotspot. At present, most scholars believe that the mRNA degradation and death between the time a good correlation exists, can be a late indicator of postmortem interval. However, in early death, mRNA degradation is not obvious, is not suitable for early postmortem interval. I believe that, mRNA stability is not high as a nucleic acid within 24h after death significant degradation does not occur this view is questionable. Because early nucleic acid determination only through immunoblotting and Rt-PCR and other semi-quantitative method, mRNA levels can not be measured even larger tiny changes. Meanwhile, earlier studies also individual differences in the elimination of the lack of rigor, research shows that different individuals within the same organization mRNA content is likely there is a big difference. For the above two points, the present study using real-time quantitative PCR detection methods that are more accurate, and through repeatedly drawn from the same individual approach to eliminate individual differences, and further study the degradation of mRNA in the case of early death, looking for its degradation and the relationship between the time of early death for early postmortem interval to provide a new means of research. The experiment was divided into three parts. The first part is from rat brain, heart, kidney extracted total RNA. Is placed after the rats were killed and 20 ℃ environment, in the afterlife Oh, 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h, 72h and 96h 11 time points for each individual in each group for 11 subjects, for each time point for all material immediately picking Extraction of total RNA. The extracted total RNA was dissolved in water 20μ1DEPC. Nano Drop 1000 using extracted RNA was measured for absorbance at 260nm and 280nm (A) value and the concentration value detected by agarose gel electrophoresis integrity. The results showed that all samples were A260/A280 ratio between 1.8 and 2.0, the concentration values ??ranging between 500ng/μl ~ 3000ng/μl. Agarose gel electrophoresis showed the integrity of all samples is good. The second part is the total RNA extracted from samples of genomic DNA (genomic DNA, gDNA) removal. Method is to use DNase I digestion of the total RNA solution, then with phenol / chloroform and inactivate DNase I revert RNA. For re-mention of the RNA was used to detect the RNA Nano Drop 1000 in absorbance at 280nm and 260nm (A) value and the concentration value detected by Rt-PCR amplification before and after removal of genomic differences. The results showed that removal of a sample of gDNA, A260/A280 ratios were between 2.0 to 2.1, the concentration values ??for the removal of a significant decrease occurred before, indicating that removal of the genome makes further purified total RNA, but also occurred in the amount of RNA losses. Rt-PCR results showed that before the removal of gDNA samples are varying degrees of gDNA of amplified bands, and after removal of impurities in the sample were not there bands. The third part is the removal of total RNA samples of gDNA P-actin mRNA concentration was determined. Method is to use real-time fluorescence quantitative Rt-PCR. Test results showed early death, brain, heart, kidney three tissues of β-actin mRNA levels were significantly decreased occurred, and within the organizations β-actin mRNA degradation and death between the time there is a good linear relationship. For previous studies of time-dependent degradation of mRNA present in the law is not precise enough determination and individual differences may exist problems, the subject for the first time design derived from the same individual repeated coverage of methods, the use is currently the most accurate real-time PCR technology, specifically studied the mRNA degradation and early relationship between the time of death, confirming the rats died early brain, heart and kidney tissue degradation of β-actin mRNA was significantly and there is a good linear relationship can be inferred through its degradation of early death time.

Estimation of Early Postmortem Interval with β-actin mRNA Degradation in Rat’s Brain, Heart and Kidney

Abstract

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