不同温度下大鼠脑组织RNA降解与早期PMI的相关性

目的探讨大鼠脑组织8种RNA指标,在不同温度下的表达水平与早期死亡时间(PMI)的相关性。方法将222只SD大鼠随机分为对照组(死后0 h)和4个实验组,实验组断颈处死后分别置于5℃、15℃、25℃和35℃的环境中,于死后1~24 h内9个时间点提取脑组织RNA,利用实时荧光定量PCR技术检测8种RNA指标(β-actin、GAPDH、RPS29、18S rRNA、5S rRNA、U6 snRNA、miRNA-9及mi RNA-125b)的表达水平,ge Norm软件选取合适内参,SPSS软件对内参标准化RNA指标进行回归分析,R软件构建推断PMI的数学模型,另选6只已知PMI的SD大鼠予以验证。结果 5S rRNA、miR-9和mi R-125b表达稳定,可作为内参指标。β-actin和GAPDH具有良好的时序性降解规律,在24 h内随PMI延长不断降解。R软件拟合得ΔCt值随PMI和温度变化的数学模型可用以推断PMI。运用β-actin和GAPDH验证模型的平均误差率分别为14.1%和22.2%。结论β-actin和GAPDH表达水平与PMI和环境温度相关性良好。本研究建立的数学模型可为温度变化条件下的早期PMI推断提供参考。

Correlation between RNA Degradation Patterns of Rat's Brain and Early PMI at Different Temperatures

Objective To explore the correlation between early postmortem interval(PMI)and eight RNA markers of rat's brain at different temperatures.Methods Total 222 SD rats were randomly divided into control group(PMI=0 h)and four experimental groups. And the rats in the experimental groups were sacrificed by cervical dislocation and respectively kept at 5 ℃, 15 ℃, 25 ℃and 35 ℃in a controlled environment chamber. The RNA was extracted from brain tissues, which was taken at 9 time points from 1 h to 24 h postmortem. The expression levels of eight markers,β-actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, were detected using real-time fluorescent quantitative PCR, respectively. Proper internal reference was selected by geNorm software. Regression analysis of normalized RNA markers was performed by SPSS software. Mathematical model for PMI es-timation was established using R software. Another 6 SD rats with known PMI were used to verify the mathematical model.Results 5S rRNA, miR-9 and miR-125b were suitable as internal reference mark-ers for their stable expression. Bothβ-actin and GAPDH had well time-dependent degradation patterns and degraded continually with prolongation of PMI in 24 h postmortem. The mathematical model of the variation ofΔCt values with PMI and temperature was set up by R software and the model could be used for PMI estimation. The average error rates of model validation usingβ-actin and GAPDH were 14.1% and 22.2%, respectively.Conclusion The expression levels ofβ-actin and GAPDH are well cor-related with PMI and environmental temperature. The mathematical model established in present study can provide references for estimating early PMI under various temperature conditions.