| Background:Postmortem interval (PMI) or the elapsed time since death refers to the time interval between the death time and the time when the corpse was found. Estimation of PMI is one of the most important and difficult problems. Since there were many influencing factors on estimation of PMI,the sole index could not provide the acute PMI in most cases. The combination research of multiple indexes and factors,that is artificial neural network(ANN) , has become the current tendency. With the development and application of computer and molecular biology,the estimation of PMI through detecting mRNA has become true. In order to provide a new method in estimation of PMI,we examined the relative content of glyceraldehyde phosphate dehydrogenase(GAPDH) andβ-actin mRNA in brain,liver,spleen and kidney of rats in this paper , using the technique of fluorescence semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).Objects:This study is designed to observe the degradation patterns of the house- keeping gene GAPDH andβ-actin mRNA of brain,liver,spleen and kidney of rats after death,to define their detection time limit in different organs,to explore the application value in estimation of PMI and to provide a new,objective and reliable observation index.Methods:56 Wistar rats were killed by cervical dislocation and placed in a chamber with temperature-controlling system at 20℃,autopsied at 0h,8h,16h,24h, 32h,40h,48h,72h,4d,5d,6d,7d,8d,9d respectively. To extract the total RNA of brain,liver,spleen and kidney according to the extraction method respectively,changes of house-keeping genes GAPDH andβ-actin mRNA were amplified and examined by one-step fluorimetric RT-PCR using GeneAmp? PCR System 9700 and 3100 Genetic Analyzer,analyzing the amplification products by GeneScan Analysis Version 3.1.Results:The amplification products of the experimental rat's GAPDH mRNA could be detected in brain within 7d,in liver within 48h,in spleen within 32h,in kidney within 40h postmortem;toβ-actin mRNA,they were 8d,72h,40h,48h respectively,and each showed a decreasing tendency regularly(its peak area became less,lightness became darker gradually and disappeared ultimately). The relative content were the ratios of the corresponding peak area and the 0h's. The degradation process of the experimental amplification products presented some regularity. Analyzing the data with software of EXCEL 2003 and SPSS 11.5,we conclude the following regression equations:1. Brain:GAPDH:y=-0.122x+0.923,R2=0.973 (x within 7d)β-actin: y=-0.104x+0.925,R2=0.973 (x within 8d)2. Liver:GAPDH:y=-0.019x+0.989,R2=0.968 (x within 48h)β-actin: y=-0.013x+0.945,R2=0.890 (x within 72h)3. Spleen:GAPDH:y=-0.025x+1.054,R2=0.969 (x within 32h)β-actin: y=-0.023x+1.021,R2=0.993 (x within 40h)4. Kidney:GAPDH:y=-0.022x+1.049,R2=0.978 (x within 40h)β-actin: y=-0.018x+0.975,R2=0.991 (x within 48h)Conclusions:We can arrive at the following conclusions from this experiment:1. The house-keeping genes mRNA of postmortem rat's have some stability.2. The stability of mRNA in different organs diverses,that is to say,every organ has its own detection time limit:to GAPDH mRNA,brain is 7d,liver is 48h,spleen is 32h and kidney is 40h postmortem respectively;toβ-actin mRNA,brain is 8d,liver is 72h,spleen is 40h and kidney is 48h postmortem respectively. GAPDH mRNA is easier to degrade thanβ-actin mRNA.3. The degradation regularity of house-keeping gene mRNA in different organs of rats detecting by one-step fluorescence semi-quantitative RT-PCR can provide a new,objective and reliable observation index for estimation of PMI and be a supplement for traditional methods.
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