实时RT-PCR检测大鼠死后管家基因mRNA的时序性降解

目的研究实时荧光定量RT—PCR方法检测死亡大鼠管家基因mRNA时序性降解的可行性，为死亡时间（Dostmortem interval，PMI）推断寻找新的研究手段。方法应用SYBR Green Ⅰ实时荧光定量RT—PCR技术．检测死后不同时间大鼠脑和脾中管家基因GAPDHmRNA及β—actinmRNA的水平，结果用循环阈值（简称Ct值）表示，分析死后经过时间与Ct值的线性关系，并建立死亡时间推断回归方程。结果GAPDH mRNA和β—actinmRNA的Ct值均与PMI之间存在显著的相关性。结论SYBR GreenⅠ实时荧光定量RT—PCR在定量分析mRNA降解的研究中是一个较理想的技术手段。选用管家基因作为PMI推断的研究对象，可在法医检案中消除其他基因因为个体差异带来的误差，更具实用性。Ct值作为动态监测机体死后不同时间点的客观指标．与死后不同时间点的线性关系良好，推断死后经过时间尤其是晚期死亡时间较为理想。

The Degradation of Housekeeping mRNA in Dead Rats by Real-time RT-PCR

Objective To explore the feasibility of real-time RT-PCR of housekeeping mRNA degradation in dead rats in order to seek new suitable techniques for post-mortem interval（PMI）. Methods The levels of GAPDH mRNA and β-actin mRNA in the brain and spleen of the rats were measured at different times after death by the SYBR Green Ⅰ real-time RT-PCR. The relative mRNA level was indicated with the Ct value, then the relationship between the Ct value and PMI were analyzed and the corresponding regression equation was obtained. Results The Ct values of GAPDH mRNA and β-actin mRNA by SYBR Green Ⅰ real time RT-PCR correlated well with the PMI. Conclusion The SYBR Green I real-time RT- PCR is a reliable technique for studying mRNA degradation. Adoption of the housekeeping genes eliminates systematical errors of other genes which have individual difference. As an objective and dynamic indication, Ct value has a good correlation with PMI, and could be applied for estimating PMI, especially in late period.