大鼠死后脑组织GAPDH mRNA多位点降解与晚期死亡时间的关系研究

The Relationship Between GAPDH mRNA Multiple Location Degradation and Advanced Stage Postmortem Intervals

目的探讨看家基因甘油醛-3-磷酸脱氢酶(GAPDH)mRNA上不同位点降解速率的规律性及其用于晚期死亡时间推断的可行性。方法选取在死后较长时间内仍能检测出扩增产物的大鼠脑顶叶皮质,对GAPDH mRNA的6个不同位点进行不同时间点(死后即刻、1d、5d、9d、12d)的实时荧光定量RT-PCR监测,这些位点依次分布于GAPDH mRNA的5′～3′端。结果用Ct值表示,并将所得数据及其比值(GAPDH mRNA1～GAPDH mRNA5与GAPDH mRNA6的比值)与死亡时间(PMI)进行线性回归分析。结果各位点GAPDH mRNA均与死后经过时间有相关性,但不同位点的mRNA降解速率不同。GAPDH mRNA1～GAPDH mRNA3的斜率为1.577、1.596、1.553,这3个位点的降解速率基本相同。GAPDH mRNA4～GAPDH mRNA6的斜率为0.936、0.892、0.829,这3个位点的降解速率基本相同。但后3个位点mRNA的降解速率要慢于前3个位点(P<0.05)。以GAPDH mRNA6作为外标,GAPDH mRNA1～GAPDH mRNA3与其的比值同PMI之间有相关性(R2=0.898,0.871,0.879),而GAPDH mRNA4和GAPDH mRNA5与其的比值同PMI之间无相关性。结论同一组织中看家基因GAPDH的不同位点存在降解速率的差异性。选取降解速率慢的靠近3′端的位点作为引物,可能更适于研究晚期死亡时间的推断。

Objective To study the degradation regularity on different sites of housekeeping gene GAPDH in the same tissue in order to deduce advanced stage postmortem interval（PMI）. Methods Rat brain cortex was collected, and 6 sites from 5′-cap to 3′-end in GAPDH mRNA sequence were measured by SYBR Green Ⅰ real-time RT-PCR to determine the degradation of GAPDH mRNA in brain cortex. Postmortem intervals were extended to 0, 1, 5, 9, 12 days. Results All the Ct values of GAPDH mRNA at different sites from the 5′-mRNA cap structure to the 3′-end （GAPDH mRNA 1-GAPDH mRNA 6） in brain correlate with the postmortem periods, while different sites showed different degradation rate. The rates of GAPDH mRNA 4-6 degradation were lower than those of GAPDH mRNA 1-3. If we used GAPDH mRNA 6 as external standard, the ratio of GAPDH mRNA 1-3/GAPDH mRNA 6 were correlated with postmortem periods. However, the relation between GAPDH mRNA 4-5/GAPDH mRNA 6 ratio and postmortem periods were not observed. Conclusion Different sites of housekeeping gene GAPDH in the same tissue share different degradation rates. The degradation of those sites near to 3′-end of mRNA show more precise to deduce advanced stage PMI.