大鼠死后管家基因mRNA稳定性及其时序性降解与PMI相关性研究

目的研究脑组织及骨骼肌内β-actin、GAPDH、RPL32、PKG1、SDHA、RPL13、HPRT、TBP、YWHAZ死后稳定性及其相对表达量与死亡时间(PMI)的相关性。方法取健康成年SD大鼠33只,依据据死亡时间不同随机分为11组(0h、1h、3h、6h、12h、24h、2d、4d、8d、16d、24d),分别在各时间点取大鼠胫骨前肌及脑组织约50mg,提取各组织总RNA,利用RT-q PCR技术检测9种管家基因的m RNA相对表达量。运用ge Norm、Norm Finder和Best Keeper软件对基因稳定性进行评价,利用spss软件对内参标准化的m RNA指标进行回归分析。结果脑组织及骨骼肌中均为HPRT稳定性最高,适合用于本研究的内参基因。脑组织中SDHA、RPL32及TBP 3种指标的相对表达量变化与PMI有一定的线性关系。骨骼肌中m RNA指标相对表达量与PMI之间没有显著的相关性。结论脑组织及骨骼肌是较适宜推断晚期死亡时间的检材。脑组织中SDHA、RPL32及TBP 3种m RNA的相对表达量变化与PMI有一定的线性关系,有望成为推断PMI的辅助指标。

The study on the stability of Housekeeping gene mRNA and the correlation between degradation and PMI in dead rats

Objective To explore the postmortem stability of nine Housekeeping gene mRNA—β-actin, GAPDH, RPL32, PKG1, SDHA, rRPL13, HPRT, TBP, YWHAZ and the correlation between its relative expression and postmortem interval (PMI) in rat brain tissue and skeletal muscle. Methods 33 healty adult SD rats were randomly divided into 11 groups at 11 postmortem interval(0h, 1h, 3h, 6h, 12h, 24h, 2d, 4d, 8d, 16d, 24d), sacrificed by cervic al dislocation and it's anterior tibial muscle and brain tissue (about 50mg) were extracted, respectively. The total RNA was extracted from organizations and the nine mRNA were detected by Real-time RT-PCR. Proper internal reference was selected by geNorm software. To evaluate the stability of genes by GeNorm, NormFinder and BestKeeper software. Regression analysis by spss software. Results The expression of HPRT is the most stable in both brain and skeletal muscle, suitable for reference gene. The relative expression levels of SDHA, RPL32 and TBP in brain tissue had a certain linear relationship with PMI. While there was no significant correlation between the relative expression of RNA and PMI in skeletal muscle. Conclusion Brain tissue and skeletal muscle could be suitable materials for extracting RNA in advanced stage PMI. The relative expression levels of SDHA, RPL32 and TBP had a certain linear relationship with PMI, which could be auxiliary indices for the estimation of PMI.