大鼠骨骼肌早期死亡时间推断的内参基因筛选

Reference genes of rat skeletal muscle for early postmortem interval estimation

目的死亡时间推断是法医病理学实践的重点。运用mRNA定量技术进行死亡时间推断已成为目前研究的热点。在进行mRNA指标挑选时,为了排除其他干扰因素的影响,实验数据必须要经过内参指标的标准化。因此,内参基因的选择对实验结果具有重要影响。方法SD大鼠颈椎脱臼处死后置于4℃、15℃、25℃、35℃环境温度下,在死后0h、6h、12h、18h、24h五个时间点取骨骼肌组织。利用实时荧光定量PCR技术测定常用内参基因Ct值,并使用三种内参评估软件(geNorm、NormFinder、BestKeeper)综合分析内参稳定性。结论综合三种内参评估软件结果,在低温条件下,RPS29、RPL13是较合适的内参,而在高温条件下,5SrRNA、18SrRNA是较合适的内参。在35℃时,GAPDH、HPRT、PGK1、UBC、B2M与死亡时间有明显相关性。

Objective Postmortem interval estimation is an important issue in the practice of forensic pathology.The application of mRNA quantitative technique for postmortem interval estimation has become matrue.In order to exclude the influence of other factors,the experimental data must be standardized by reference genes in the selection of mRNA markers.Therefore,the selection of reference genes has an important influence on the experimental results.Methods SD rats were executed by cervical dislocation and were placed at ambient temperatures of 4℃,15℃,25℃and 35℃.Skeletal Muscle were collected 0 h,6 h,12 h,18 h and 24 h after death.The cycle threshold values of commonly used reference genes were measured by real time quantitative PCR,and the stability of reference genes was comprehensively analyzed by three evaluation software(geNorm,NormFinder and BestKeeper).Conclusions Integrated the results of three software,RPS29 and RPL13 are more stable under low ambient temperatures.While under high ambient temperature,5SrRNA and 18SrRNA are more appropriate as reference genes.At 35℃,GAPDH,HPRT,PGK1,UBC and B2M has obvious correlation with the postmortem interval.