| Postmortem interval means time interval from the finding and checking of the body to the time of death. The exact estimation of postmortem interval has important practical value in forensic science. The exact estimation of PMI becomes an important and difficult question in forensic science. Traditional methods have been investigated to define the PMI in autopsy cases, however, these methods are sketchy, and they are subject to environmental impact and cause of death. With the development of molecular biology, the time-dependent degradation of nucleic acid has become a focus of attention in forensic science.RT-qPCR has been widely used in PMI estimation to quantify the RNA transcript level with great precision and efficiency, while correct normalization of quantitative gene data is always a critical step to obtaining reliable results, especially for accurate PMI estimations. However, recent studies were limited to consider seperate factor for model building, such as single tissue, temperature, casue of death and so on. Thus, the actual PMI estimation was more or less limited without consideration of multi-factor.In this study, RT-qPCR was performed to detect the transcript levels of RNA makers in both human and rat tissues (myocardium, liver and skin) to find optimum reference markers which were less affected by the PMI and other factors. Subsequently, target RNAs that exhibited transcript abundance changes associated with PMI were applied to establish mathematical models for PMI determination with different tissues and temperatures. Rat and human tissues with specific PMIs were used to validate the accuracy and reliability of these mathematical models and to figure out the influence of temperature, cause of death and other factors. The purpose is to provide a new means and biology targets for estimating PMI. Moreover, qRT-PCR was applied to observe the degradation patterns of variety RNAs in rat’s heart with PMI of different causes of death.Objection1. To find accurate and reliable reference RNA markers for precise PMI estimation in both human and rat tissues by RT-qPCR.2. Four continued temperature groups were set up to figure out the relationship between RNA degradation and postmoretem. Then, normalized RT-qPCR data were used to constructe multi-parametric mathematical model for PMI estimation.Take ΔCt values of rat and human specimens (with known PMI) to verify mathematical model and calculate error rate between estimated PMI and real PMI to figure out the influence of temperature, cause of death and other factors for PMI estimation.3. SD rats were randomly divided into 3 groups and killed by cervical amputation, mechanical asphyxia and hemorrhagic shock. qRT-PCR was applied to observe the degradation patterns of variety RNAs in rat’s heart with PMI of different causes of death. The specific relationships between relatively expression of each kind RNA and PMI can be used as opportune indicators to estimate the exact PMI of different causes of death.Methods and resultsPart 11. Three tissues (myocardium, liver and skin) were collected from 79 individuals obtained by autopsy at the Institute of Criminal Scientific Technology in Shanghai. Postmortem interval, temperature, cause of death and other parameters were clear of each case. Samples were immediately transferred to RNAlater and then stored at-80℃ till extraction of total RNA.2. Sprague-Dawley rats (n=288) were sacrificed by cervical dislocation and were randomly divided into four groups. The bodies of each group were respectively kept at 5±1℃,15±1℃,25±1℃ and 35±1℃ in a controlled environment chamber. The myocardium, liver and skin tissues were extracted at Oh, 1h,3h,6h,12h,24h,36h, 48h,72h,96h,120h and 144h after death in each group. These rats were used to develop models for estimating the PMI. Moreover, six rats were sacrificed in the same way and were randomly divided into two temperature groups (10±1℃ and 20±1℃) to validate the models, meanwhile three samples from each group were taken at PMIs of 15h,45h and 95h. All the samples were immediately transferred to RNAlater and then stored at -80℃ till extraction of total RNA. Twelve usually used endogenous biomarkers, ACTB, GAPDH,18S, RPS29,5S, U6, Let-7a, miR-1,miR-133a, miR-206, miR-122 and miR-203, were chosen and primers were designed for this experiment, respectively.3. Total RNA was isolation by a modified TRIzol reagent; the concentrations and purity of total RNA; cDNA synthesis; and Ct values of RNA were analyzed by the qRT-PCR.4. The stability analysis of twelve chosed reference biological markers was performed using geNorm. After unify the internal genes of human and rats, the results indicated 5S, U6, miR-133a and RPS29 were selected to be endogenous reference genes in postmortem myocardium tissues, while 5S, U6, RPS29 and miR-122 were selected as reference genes in postmortem liver tissues and 5S, U6, RPS29 and miR-203 were selected as reference genes in postmortem skin tissues.Part 25. A curve estimation analysis between normalized ΔCt values of ACTB and GAPDH and PMI was performed with three mathematical functions (linear, quadratic and cubic).6. ΔCt values of rat and human specimens (with known PMI) were taken into mathematical model and calculate error rate between estimated PMI and real PMI to evaluate the impact of figure out the influence of temperature, cause of death and other factors for precise PMI estimation. The low error rate (14.4±2.2% of rat validation samples,34±19% of human case 1-69) of present study demonstrate the reliability of this mathematical models.7. The degradation rate of RNA markers are accelerated under higher temperature. Since the the postmortem RNA degradation is significantly affected by temperature, the average temperature is essential for PMI estimation. The exact record of average temperature will improve the accuracy of PMI determination effectively.8. Among three tissues, error rates of liver tissue sare lowest and liver tissues are more appropriate for the estimation of the early PMI. On the contrary, myocardium tissues are more appropriate for the estimation of the late PMI. The error rates of skin tissues are generally larger than others and the skin tissues seemed to most unstable against PMI or temperature. But in some special cases (such as the case of dismembered body), the skin may be the only existing samples, so the research of skin tissues would be meaningful.9. The causes of death also have influence on RNA transcript quantities in this study. The error rate of GAPDH was negative in hemorrhagic shock and mechanical asphyxia group, while error rate was positive in brain trauma groups, which confirmed that the GAPDH mRNA transcript levels were significantly influenced by cause of death. GAPDH expression up regulated in the death of hemorrhagic shock and mechanical asphyxia thus estimated PMI of these decedents were lower than its real PMI.Part 310. Sprague-Dawley rats (n=135) were randomly divided into 3 groups and killed by cervical amputation, mechanical asphyxia and hemorrhagic shock, the bodies were kept at 25±1℃ in a control chamber. Total RNA was extracted from myocardium at Oh,3h,6h,12h,24h,36h,48h,72h and 96h after death in each group.All the samples were immediately transferred to RNAlater and then stored at -80℃ till extraction of total RNA. Quantitative real time PCR was used to detect the expression amounts of GAPDH, ACTB, HIF-1, iNOS, TNF-α, IL-6 mRNAs and U6 snRNA by calculating Ct values.11. The GAPDH, HIF1-α, iNOS, TNF-α and IL-6 expression quantities varied by cause of death in the early PMI, then the expression quantities began to decline gradually with longer PMI due to degradation of all mRNAs. The specific relationships between relatively expression of each kind RNA and PMI can be used as opportune indicators to estimate the exact PMI of different causes of death.Conclusions and outlookWith the assistance standardized process of qRT-PCR, stable internal reference genes were selected in present study. Furthermore, we established a mathematical model with multi temperature, tissues and markers to estimate the PMI. After validation by rat samples and real human cases, the low error rate of present study demonstrate the reliability and accuracy of this mathematical models and evaluate the influence of multi-parameters. This study will continue to collect human samples to validate and then modify the mathematical model, which represents a new progress to increase the accuracy of PMI estimation in forensic application. |
PDF Full Text Request