| Objective To study the relationship between the relative expression levels of 3kinds of RNA and postmortem interval?postmortem interval,PMI?in brain and liver tissues in rats,and to compare the applicability of the two tissues in the estimation of PMI,and to provide a theoretical basis for the application of RNA in the estimation of PMI.Methods 48 Wistar rats were randomly divided into 6 groups,and all of them were killed by neck dislocation.Then they were placed in the environment of altitude2260 m,room temperature 12±1 ? and humidity 40±2%.At the postmortem 0 h,24 h,48 h,72 h,120 h,168 h,cerebral cortex and right hepatic lobe were taken.A part of brain and liver tissues were fixed with 4% paraformaldehyde and then observed by HE staining.Then the total RNA of the other part of brain and liver tissues were extracted,and the cyclic thresholds?Ct values?of miRNA?brain specific miR-124,liver specific miR-122?and glyceraldehyde-3-phosphate dehydrogenase?GAPDH?,beta-actin??-actin?m RNA in the two tissues were detected by real-time fluorescence quantitative PCR?RT-q PCR?.The internal reference was selected by Best Keeper program,and the Ct value of target RNA was converted into 2-??Ct.Single factor variance analysis was used to examine 2-??Ct by SPSS and Graph Pad software.The curve regression analysis was used to examine?Ct value.And regression models were constructed between ?Ct value and PMI.Results 1.The standard deviation?standard deviation,SD?and coefficient of variation?coefficient of variation,CV?of miR-124 in brain tissue are the smallest,and the SD and CV of miR-122 in liver tissue are the smallest.2.The relative expression levels 2-??Ct of GAPDH and?-actin m RNA in brain tissue increase gradually within the postmortem 72 h,and the peaks are reached at the postmortem 72 h,after the postmortem 72 h the relative expression levels decrease gradually?P<0.05?.The relative expression levels2-??Ct of hepatic GAPDH and?-actin m RNA are unchanged within the postmortem 24 h,and decrease gradually after the postmortem 48 h?P<0.05?.3.The optimal regression models between ?Ct values of all target RNA molecules and PMI are the cubic model.The correlation coefficient r of liver tissue is larger than that of the brain tissue.The regression equations of the optimal models are as follows:yGAPDH m RNA of brain=3.280-0.028x+2.343×10-4x2-5.440×10-7x3,y?-actin m RNA of brain=4.231-0.039x+3.985×10-4x2-1.103×10-6x3,yG APDH m RNA of liver=4.965-4.460×10-3x+4.328×10-4x2-1.666×10-6x3,y?-actin m RNA of liver=5.660-0.019x+6.064×10-4x2-1.947×10-6x3.Conclusion 1.MiR-124 and miR-122 can be used as internal reference in brain and liver tissues respectively by the internal reference selection.2.In the environment of altitude 2260 m,room temperature 12±1 ? and humidity40±2%,the relative expression levels 2-??Ct of GAPDH and ?-actin m RNA in brain tissue of rats increase gradually within the postmortem 72 h,and the peaks are reached at the postmortem 72 h,after the postmortem 72 h the relative expression levels decrease gradually.The relative expression levels 2-??Ct of hepatic GAPDH and ?-actin m RNA are unchanged within the postmortem 24 h,and decrease gradually after the postmortem 48 h.The liver tissue is more suitable for the estimation of PMI than the brain tissue.3.In the environment of altitude 2260 m,room temperature 12±1 ? and humidity40 ± 2%,the optimal regression models between ?Ct values of GAPDH and ?-actin m RNA and postmortem interval in brain and liver tissues of rats are the cubic models. |
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