不同种类RNA作为TSD推断内参基因的研究筛选和评估  

Screening and evaluating the reference genes of different types of RNA for estimation of time since deposition

作　　者：郭顺天 郑刘霞 孙启凡 赵一霞 季安全 叶健 陈雯莉[1] 胡胜 Guo Shuntian;Zheng Liuxia;Sun Qifan;Zhao Yixia;Ji Anquan;Ye Jian;Chen Wenli;Hu Sheng(College of life science and technology of Huazhong Agricultural University,Wuhan 430070;Institute of Forensic Science of China&National Engineering Laboratory for Forensic Science,MPS Key Laboratory of Forensic Genetics,Beijing 100038,China)

机构地区：[1]华中农业大学生命科学技术学院,湖北武汉430070 [2]公安部鉴定中心,现场物证溯源技术国家工程实验室,法医遗传学公安部重点实验室,北京100038

出　　处：《中国法医学杂志》2023年第4期413-419,424,共8页Chinese Journal of Forensic Medicine

基　　金：国家重点研发计划项目(2022YFC3341002);公安部鉴定中心基本科研业务费项目(2022JB021)。

摘　　要：目的准确估算血迹离体时间能为刑事案件调查提供信息或线索。本文利用qPCR技术检测血迹中RNA的降解情况,进而筛选并评估适用于血迹离体时间推断的内参基因(reference genes)。方法本研究制备10名志愿者0~90 d区间范围内7个时间节点共70份样本的总RNA为实验材料,基于其中6名志愿者的样本的qPCR数据,使用geNorm和NormFinder算法对14个候选内参基因(let-7g-5p,let-7i-5p,miR-191-5p,miR-484,miR-103a-5p,miR-423-5p,PPIA,ACTB,5SrRNA,18SrRNA,miR-93-5p,U6b,SNORD24,SNORD38B)进行稳定性评估,筛选内参基因及其使用方案。选取全部10名志愿者70份样本,验证内参基因及其使用方案的校正效果。结果qPCR检测显示miRNA在血迹离体后90 d范围内表现出的稳定性较强。综合使用geNorm和NormFinder两种算法获得的内参基因使用方案为:以let-7g-5p为单基因内参,或以let-7g-5p、U6b和miR-191-5p组合为三内参基因。70个样本对两个内参基因使用方案的校正能力验证显示,多基因组合内参方案的三次方程模型能更准确推断血液斑迹的离体时间。结论本研究首先推荐以ACTB为标记,以let-7g-5p、U6b、miR-191-5p组合的三内参基因三次方程校正模型用于血迹离体时间推断检验鉴定技术研发;在考虑提高检验效率、简化实验流程等需求而需要选择单基因作为内参基因时,可以选择ACTB为标记,使用let-7g-5p为内参基因的三次方校正模型。Objective Accurately estimating the time since deposition(TSD)of blood stains can provide information or clues to the investigation of criminal cases.In this study,qPCR was used to detect the degradation of RNA in blood stains.And then,screening and verification of reference genes that are suitable for estimation of blood stains TSD was conducted.Methods A total of 70 samples from 10 volunteers within 0-90 days were prepared as experimental materials in this study.Based on the qPCR data of the samples from 6 of the volunteers,geNorm and NormFinder procedures were used to evaluate the stability of 14 candidate reference genes(let-7g-5p,let-7i-5p,miR-191-5p,miR-484,miR-103a-5p,miR-423-5p,PPIA,ACTB,5SrRNA,18SrRNA,miR-93-5p,U6b,SNORD24,SNORD38B),and to obtain the optimal reference genes or genes combination.70 samples from all 10 volunteers were used to verify the correction effect of internal reference genes or gene combinations.Results The qPCR detection showed that miRNA was stable within 90 days since deposition of blood stains.The internal reference gene obtained by combining geNorm and NormFinder procedures is as follows:let-7g-5p as a single reference gene,or let-7g-5p,U6b and miR-191-5p as a combination of three reference genes.Verification of the correction ability of reference gene usage schemes for 70 samples showed that the cubic equation model of multigene combination reference genes was more accurate in estimating the blood stains TSD.Conclusion Firstly,this study recommended the use of ACTB as the marker,and let-7g-5p,U6b,and miR-191-5p combination of three reference genes cubic equation correction model for estimating the blood stains TSD.In order to improve the detection efficiency and simplify the experimental process,ACTB can be selected as the marker and let-7g-5p as the single internal reference gene of the cubic correction model.

关 键 词：法庭科学 内参基因 qPCR 核糖核酸(RNA) 离体时间(TSD) 

分 类 号：D919.2[医药卫生—法医学]