不同死亡原因大鼠脑组织5种RNA降解与早期PMI的相关性

The relationship between the degradation of five RNAs and early PMI in brain tissue of rats with different causes of death

目的建立不同死亡原因下早期PMI与RNA△Cq值的数学模型,为死因变化条件下的早期PMI推断提供参考。方法取SPF级成年雄性SD大鼠30只,随机分为5组(颈动脉大失血致死、勒死、溺死、服有机磷农药致死、CO中毒致死),每组6只。处死后大鼠置于恒温恒湿箱(20℃,55%湿度),死后0~24 h内7个时间点分别取脑组织RNA。测定每个时间点5种RNA(β-actin、GAPDH、miR-9、miR-124、miR-125b)Cq值,通过QBase+V(3.4)软件geNorm程序选择内参,运用SAS 9.4软件分析早期PMI与RNA△Cq值的相关性,并得到数学模型。另取SPF级成年雄性SD大鼠15只,随机分为5组,分别在死后10 h、20 h取材进行反推验证。结果(1)miR-124在本实验体系中无法捕捉荧光,不能作为本实验体系的候选基因;(2)不同致死原因下大鼠脑组织β-actin、GAPDH、miR-9、miR-125b四种基因稳定性不同,SAS 9.4软件分析早期PMI与RNA△Cq值的相关性,得到数学模型可用于早期PMI推断。结论(1)死因会影响大鼠死后脑组织RNA的降解,不同死因应选用不同的内参基因;(2)本实验体系下建立的数学模型可为不同死亡原因致死后早期PMI推断提供参考。

Objective To establish a mathematical model of early PMI and RNA△Cq under different causes of death,which provides a reference for early PMI inference under the condition of changing causes of death.Methods Thirty adult male SD rats with SPF level were randomly divided into 5 groups with 6 animals in each group(Death by carotid artery major hemorrhage;Death by strangle;Death by drown;Death by organophosphorus pesticides;Death by CO poisoning).The rats were placed in a constant temperature and humidity incubator at 20℃and 55%humidity after death.RNA was extracted from brain tissue at 7 time points within 0~24 h postmortem.The Cq values of five RNA(β-actin,GAPDH,miR-9,miR-124,miR-125b)were measured at each time point.QBase+V(3.4)software geNorm program was used to select the internal parameters.The correlation between early PMI and RNA△Cq was analyzed by SAS 9.4 software,and a mathematical model was established.Another 15 adult male SD rats with SPF level were randomly divided into 5 groups.The RNA from brain tissue were collected at 10 h and 20 h after death for back-inference verification.Results(1)The fluorescence of miR-124 could not be captured in this experimental system,and this gene could not be used as a candidate gene in this experimental system.(2)The stability of the four genes(β-actin,GAPDH,miR-9 and miR-125b)in rat brain tissue varied under different conditions of death.Using SAS 9.4 software,the correlation analysis were conducted between early postmortem interval(PMI)and RNA△Cq values,and the mathematical models were set up for early PMI estimation.Conclusion(1)The causes of death can affect the degradation of RNA in postmortem brain tissue of rats,and different reference genes should be selected under different causes of death.(2)The mathematical model established under this experimental system can provide reference for early postmortem PMI inference of different causes of death.