Estimation Of Early Postmortem Interval With β-actin MRNA Degradation In Rat's Brain, Heart And Kidney

Estimation of time of death is always the hotspot and difficulty of forensic pathology. Time of death (which is also called Postmortem interval, PMI) is the time interval since death. PMI is divided into early PMI and the long-term PMI according to the changes of body and demands of forensic practice, early PMI is limited to 24h since death. Obviously, estimation exactitude of early PMI is more significant for the case inspection than advanced stage PMI. However, actual estimation of early PMI can only depend on some early postmortem phenomena (algor mortis, livor mortis, rigor mortis and supervital reaction,etc) and digestion degree of gastric content, which are rough, experiential and subjective methods.In the laboratory research context, study on estimation of PMI refers several disciplines and fields, such as chemical methods(blood measurement, ionic concentration change measurement of vitreous humor),immunohistochemistry methods (examining the change of tissue modality),photogrammetry methods(CT,MRI,MRS) and molecular biology methods(study time-depend degradation of nucleic acid and protein), among them, in the molecular biology field, it is a hotspot to use time-depend degradation of nucleic acid (especially mRNA) to estimate PMI. Now it is widely agreed that mRNA degradation is distinctly correlated with PMI, which make the mRNA degradation to be a good index for estimation of advanced stage PMI. However, mRNA is not suitable for estimation of early PMI because it has no distinct degradation at the early death.As far as the author concerned, as a kind of instable nucleic acid, it is suspectable that mRNA was thought to have no distinct degradation during the 24h since death. A little change of mRNA, even for greater change could not be measured at early time except some half-quantitative methods such as northern blot and Rt-PCR. Meanwhile, early research is not so precise to eliminate individual difference. It is indicated that there maybe different mRNA content in the same tissue from different individuals.So as a more precise measurement method, real-time quantitative RT-PCR will be used in this experiment. More research on mRNA degradation and its relationship with early PMI will be did through reduplicative extraction from same individual, which could afford a new way for estimation of early PMI.The experiment contains three parts. The first part is to extract total RNA from rat's brain, heart and kidney. Rats were killed and kept with a constant temperature of 20C.Extract 11 times from each individual in every group at 0h,4h,8h,12h,16h,20h,24h.36h,72h,96h after death. Dissolve total RNA with 20μl DEPC water and measure its absorbency and concentration at 260nm and 280 nm with Nano Drop 1000, then check its integrality with agarose gel electrophoresis. Results show that A260 and A280 of all the samples are within the range of 1.8-2.0, while their concentrations are between 500 ng/μl to 3000 ng/μl, and the integrality of all samples are qualified.The second part is to remove genomic DNA by digesting total RNA solution with DNase I and then inactivating DNase I with phenol/chloroform to re-extract RNA. Then measure its absorbency and concentration at 260nm and 280 nm with Nano Drop 1000, examine amplified difference between with and without gDNA with Rt-PCR. Results show that A260/A280 of samples with gDNA removed are 2.0-2.1, but concentrations have obviously decreased, which indicate removing gDNA could purify total RNA, but cause RNA losses at the same time. Rt-PCR results show there are varying gDNA amplified bands on samples whose genome have been not removed.The third part is to use real-time quantitative RT-PCR to measureβ-actin mRNA concentrations of total RNA samples with gDNA removed. Results show that sharp decreases inβ-actin mRNA level were observed in all the mentioned three tissues and perfect linear relationships between the degradation ofβ-actin mRNA and PMI were discovered.In consideration of the lack of enough preciseness and the possible individual differences in the study of the time-dependent degradation of mRNA within early PMI, we designed the method of sampling from the same individual at different PMIs and applied the quantitative method of real time quantitative PCR, which is the most accurate method to date. By taking these measures, we confirmed the presence of obvious degradations ofβ-actin mRNA within rat's brain, heart and kidney in short-term time interval after death, as well as the possibility of estimating early PMI throughβ-actin mRNA degradation in these tissues.