Estimation Of Postmortem Interval Using MicroRNA And 18S RRNA Degradation In Rat Cardiac Muscle

Postmortem interval, also as known as time of death, means time interval from the found and check of the body to the time of the 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.There are many methods for the estimation of PMI, mainly based on the traditional body of phenomena (such as livor mortis, algor mortis, rigor mortis) and the digestive process of gastric contentS,fly larvae growth law, and so on. But these methods are sketchy, and they are subject to environmental and geographical impact. Recent years, with the development of molecular biology, the nucleic acid in the body after death, its time-dependent degradation was gradually applied the research of postmortem interval.Many scholars have found there is a good linear relationship between the degradation of housekeeping genes and the postmortem interval, and it is suitable for use in estimation of PMI. The research about RNA has focused on two common housekeeping genes:GAPDH andβ-actin. However, GAPDH andβ-actin belong to high abundance mRNA in the body, and in the early time of death it's curve is not obvious, more suitable for internal reference. At the same time, the degradation of mRNA is also affected by environmental factors, which limits its application in estimation of PMI. We have found that small RNA(microRNA) and 18S ribosomal RNA, had good stability and variation in rats at different stages of death, and they would be more suitable for estimation of postmortem interval.In preliminary experiments, we have found that in rat cardiac cells some nucleic acids such as miRNA and 18S rRNA have certain law of degradation and they are less affected by environmental factors after death. We intend to constant environmental temperature and humidity, and study rat myocardial tissue, evaluate the value of changes of miRNA and 18S rRNA in the estimation of PMI using real time fluorescence quantitative PCR. The purpose is to provide a new means and biology targets.PartⅠExtraction of Rat Heart and Myocardial Morphological IdentificationObjective:To obtain rat cardiac tissue, and observe the change of myocardial morphology in differernt time of death.Methods:SD rats were sacrificed by cervical dislocation and placed at ambient temperature 25℃with a humidity 50%, and dissected at different times after death. Rats myocardial tissues were obtained and fixed in formalin for HE staining.Results:Successfully obtained rat myocardium of different PMI. Myocardial HE staining showed that the changes of myocardial autolysis are the same with the traditional postmortem phenomena development. After 24 hours, a small amount of myocardial cells began to autolyze, and cell began to rupture; after 96 hours, most of cells autolyzed, and cell structure was unclear, nuclear was disappeared; after 144 hours, the striated muscle completely disappeared, and myocardial cells were completely collapse and fragmentation; after 168 hours, the cells were no shape, no nuleus.PartⅡExtraction of Rat Myocardial tissue and analysis of RNA degradationObjective:To research the relationship between the degradation of RNA and postmortem interval.Methods:Extracted RNA using Trizol, and detected the concentration and purity of RNA using NanoDrop 1000. Observed changes of the RNA degradation of different postmortem interval using non-denaturing gel electrophoresis.Results:The gel electrophoresis showed the luminance of 28S band was discending with postmortem interval, and disappeared after 24 hours, the luminance of 5S band did not change significantly. After 48 hours, RNA was degraded into small fragments. PartⅢAnalysis of microRNA and 18S rRNA of Rat myocardial tissue using real-time fluorescence quantitative PCRObjective:To research the relationship between the change of microRNA and 18S rRNA in rat myocardial tissue and postmortem interval.Methods:Detected the content of microRNA and 18S rRNA in rat myocardial cells with different postmortem interval using real-time fluorescence quantitative polymerase chain reaction.Results:The miRNA(like mir-1-2) level in rat myocardial tissue showed no significant changes within 120 hours after death, and then began to decline. The 18S rRNA level increased gradually within 96 hours after death, and then declined slowly. The nonlinear relationship were established between Ct value (18S rRNA),△Ct value (difference between 18S rRNA and mir-1-2) and PMI. The regression equations were Y1=0.0012X2-0.1759X+19.022; Y2=-0.0006X2 +0.1192X-0.1833. The R2 of conics fitting were 0.9487 and 0.8072.Part IV Verification of human myocardial tissue samples whose PMI is already knownObjective:To initially verify the relationship between the human myocardial samples'microRNA and 18S rRNA level whose PMI was exactly known already and the time of death known from the experiments.Methods:Detect the level of miRNA and 18S rRNA in human cardiac cells using real-time fluorescence quantitative polymerase chain reaction. Compare the results obtained from the regression equation with the exact known PMI, and value the error between them.Results:The level of microRNA and 18S rRNA in human cardiac muscle was rich than rat. Put the data substitute into the regression equation and obtained the results, showed that by rectification it was basic same with the actual PMI, and the error was in 2 hours. Estimation of Postmortem Interval is always an important and difficult problem in forensic science. The biology marker research used for estimation of postmortem interval mainly focused on DNA, and using streaming technology, Feulgen staining, comet assay and other methods to analyze the content of DNA downward trend after death. And analyze the degradation of housekeeping genes mRNA, such as GAPDH andβ-actin after death. However, GAPDH andβ-actin belong to high abundance mRNA in the body, and in the early time of death it's curve is not obvious, more suitable for internal reference. Selecting one or more indicators of PMI is the key to break through the traditional comprehensive determine postmortem interval. Our study have found that miRNA and 18S rRNA had good stability and regularity in the estimation of PMI, and they may have a good value in the postmortem interval in forensic science.Our study had found that the miRNA was stable relatively, using ABI Fast Real-time PCR instrument. After death, within 120 hours, the content of the miRNA in the body was in a relatively stable level, and did not change with the change of the PMI, and it began to decline after 120 hours. This is because the non-coding microRNA is a type of single-stranded small RNA, only 21-25 nucleotides. Because the fragment is very small, it is very stable in vivo. So, in the early hours after death, miRNA can be used as a very stable internal reference to reflect other indicators. Therefore, miRNA as a molecularmarker because of its stability in the postmortem interval has a very important value.The study also found that the content of 18S rRNA gradually increased in the early stage after the death of rats, peaking at around 96 hours, and the began to decline. Through analysis, we consider that the main of 18S rRNA present in the ribosome by a number of ribosomal protein complex composed of complexes, and only few present separately. This provides a relatively safe environment for 18S rRNA, and can isolate RNA enzymes and other chemical factors, let 18S rRNA remain stable. As the time of death, protein degradation, 18S rRNA is released from the complex, and within a stage after death, the content increased, with a certain regularity. Within 96 hours after death, 18S rRNA degradation rate is lower than its release rate from the protein complexes.; and after 96 hours, because most of the 18S rRNA have been released, the degradation rate began to exceed the released rate, that caused the content of 18S rRNA decreased gradually.We consider that, the comprehensive utilization of miRNA stability and regularity of 18S ribosomal RNA to infer postmortem interval, is more accurate and reliable than the results of single use of housekeeping genes with individual semi-quantitative PCR. Since the occurrence and development of the postmortem phenomena are restricted by the various conditions, regardless of the use of DNA,RNA or protein degradation, there are certain defects and deficiencies to estimate postmortem interval. And using one method or biomarker is difficult to accurately estimate postmortem interval. How to select and optimize the different indicators and the comprehensive application of these indicators and methods is a research direction in forensic science using biological macromolecules to estimate the PMI in the future.Our study found that the relationship is close between 18S rRNA and the PMI (Y1=0.0012X2-0.1759X+19.022), using more accurate method—real time PCR, and the concentration of miRNA remained stable in a longer period of time after death. By collecting human tissue samples whose PMI is already known, and preliminary experiment analysis, there are some errors between the results and the actual time of death, but the error is in the 1-2 hours. So our experiment demonstrated that the changes of microRNA and 18S rRNA could be more accurate to estimate the postmortem interval.