Screening And Application Of MRNA Markers In Wound Age And Postmortem Interval Estimation

Objective:Based on screening the differential genes involved in rat skeletal muscle damage,to detect the expression level of 9 mRNA(Dclre1b,Asb5,Fam210 a,Lin37,Hs6st1,Lrrc41,Ier3,Leprot,Gpnmb)in muscle of rats with different wound age and postmortem interval applying real time PCR(q PCR)technique,and to analyze the correlation between these genes,wound age and postmortem interval.The representative gene markers were identified for estimating wound age and postmortem interval in rat skeletal muscle,and he mathematical models were built for estimating wound age and postmortem interval.These methods may provide advanced strategies for gene markers screening in forensic research.Methods:A total of 168 Sprague–Dawley rats were randomly divided to control and contusion groups(0,4,8,12,16,20 and 24 h,n=24).Contusion groups were divided to 3postmortem interval subgroups(0,12 and 24 h,n=8).Rats were anesthetized by 3%pentobarbital sodium(0.13 m L /100 g).A 500 g counterpoise was allowed to fall freely30 cm onto the quadriceps femoris of right posterior limbs.After killed off,24 rats were stored in climate chamber(16℃)for 0h,12 h and 24 h respectively,with 8 rats at each postmortem time point.100 mg muscle sample was dissected from the injured quadriceps femoris of the right posterior limb and frozen in liquid nitrogen.q PCR was used to detect the mRNA expression level of Dclre1 b,Asb5,Fam210 a,Lin37,Hs6st1,Lrrc41,Ier3,Leprot and Gpnmb in different wound age and postmortem interval.Pearson correlation analysis was used to evaluate the correlation of wound age,postmortem interval and target mRNA.Moreover,partial least squares discriminant analysis(PLS-DA)was used to preliminarily classify wound age and postmortem interval after screening indicators by SIMCA-P 14.1.Then,Fisher Discriminant Analysis models of wound age and postmortem were established interval.Leave-One-Out Cross Validation is used to verify the accuracy of models.Results:(1)All of the OD260/OD280 values of total RNA samples were between 1.8 and 2.0,and the RIN values were greater than 7.0.The amplification efficiency of 9 target genes and 2 reference genes was between 90% and 110%,and the correlation coefficients were all greater than 0.990,indicating that the amplification efficiency of each gene was high and the reaction system was appropriate,which was suitable for subsequent analysis.(2)Compared with the control group,the change trends and degrees of the relative expression levels of the 9 genes were diverse in different wound age and postmortem interval.(3)Pearson correlation analysis was performed with SPSS to evaluate the correlation between wound age and postmortem interval in 9 indicators,where 4indicators were correlated wound age and 5 indicators were correlated postmortem interval significantly.(4)The PLS-DA results showed significant differences among the4 time periods(0h,4h,8-12 h and 16-24h)after injury when 4 wound age correlated indicators were inputted,and significant differences among the 3 death groups when 5postmortem interval correlated indicators were inputted.(5)The FDA model established according to the contusion groups of 0h,4h,8-12 h and 16-24 h was able to correctly classify 94.6% of the original grouped samples,and the accuracy of internal cross validation was 92.8%.The FDA model established according to the 0h,12 h and 24 h death groups could correctly classify 89.6% of the original grouped samples,and the accuracy of internal cross validation was 89.6%,which could well distinguish postmortem interval in three different groups.Conclusion:In this study,we analyzed the differential expression of 9 mRNA of rat skeletal muscle at different wound age and postmortem interval.The results showed that the detection indexes changed over wound age and postmortem interval.Highly wound age or postmortem interval correlated indicators were screened by Pearson correlation analysis,which provided a research basis for the establishment of wound age and postmortem interval indexes screening system.Based on the index screening,PLS-DA model and Fisher discriminant model are established,which can precisely predict the wound age and postmortem interval,and verify the validity of the selected indexes,which provided a new idea for the subsequent research.