Associativity Study Between Housekeeping Genes MRNA Degradation Regularity In Dead Rats And Advanced Stage Postmortem Intervals

Objective In order to explore ideal tissue for determination of the late time of death, and establish linear regression equation between mRNA degradation and postmortem intervals, the rate of mRNA degradation from housekeeping gene GAPDH andβ-actin investigated. One step RT-PCR technique and Real time RT-PCR technique are applied in evaluating the correlation between rate of mRNA degradation in SD rats and postmortem intervals, especially in advanced stage PMI.Methods (1) A total of 28 SD rats were grouped and left in artificial climate box at 20℃andhumidity 50% , day and night each for 12 hours. Each 4 rats were killed and corpses were keptin artificial climate box for 0, 1,3, 5, 7, 9, 12 days. The heart, liver, spleen, lung, kidney andbrain were stored at -80℃immediately after frozen in liquid nitrogen. (2) Total RNA wasextracted from each sample using TRIzol reagent (Invitrogen, USA) and SV Total RNA IsolationSystem (Promega, USA) according to the manufacturer' s instruction. Concentration and yieldsof total RNA in the extracts were measured. (3) Total RNA was reverse transcribed usingOne-Step RT-PCR Kit (Takara, Japan) according to the manufacturer' s instruction. Integraloptical density (IOD), Relative grayscale (RG) were measured by the image analysis in the gelimage analytical system. Spleen and brain were picked out to use for real time RT-PCR. (4) Thetarget gene mRNAs were detected by real-time PCR from the samples,which kept in artificialclamite box up to 12 days. By this methods, the relative mRNA level was indicated as the C_tvalue, the C_t value correlates inversely to amount of target mRNA in the sample. Thecorresponding regression equation was obtained. (5) In order to observe degradation regularity ofdifferent sites in the same housekeeping gene GAPDH, SYBR Green I real-time RT-PCR wasused to measure GAPDH mRNA from the 5' -mRNA cap structure to the 3' -end in intactmRNA by 6 pair of primers. Postmortem intervals were extended to 0, 1, 5, 9, 12 days.Results (1) The results showed that IOD and RG of amplification products by one stepRT-PCR in GAPDH andβ-actin mRNA decreased over time with the significant correlation ofpostmortem intervals. Amplification products of two housekeeping genes were detected at fivedays postmortem in the spleen and brain, three days postmortem in the heart and kidney, one daypostmortem in the liver and lung of rats. (3) The results showed that the C_t values of GAPDHmRNA andβ-actin mRNA by SYBR Green 1 real time RT-PCR correlates with the postmortemperiods. Thus, we got the equations of correlation between rate of mRNA degradation and postmortem periods. The equations are as follows:brain GAPDH: Y = 15.312+ 1.53 1X ,R~2=0.943brainβ-actin: Y = 15.609 + 1.750X ,R~2=0.953spleen GAPDH: Y = 19.571+1.453X , R~2=0.852spleenβ-actin: Y = 21.769+1.274X ,R~2=0.808(4) The C_t values of GAPDH mRNA at different sites from the 5' -mRNA cap structure to the 3 ' -end (GAPDH mRNA 1- GAPDH mRNA6) in brain all correlates with the postmortem periods, while different sites share the different degradation rate. The linear regression equations are as follows:GAPDH mRNA1: Y = 15.501 + 1.577X R~2=0.968, slope rate was 1.577GAPDH mRNA2: Y = 15.717+ 1.596X R~2=0.980, slope rate was 1.596GAPDH mRNA3: Y =15.772 +1.553X R~2=0.973, slope rate was 1.553GAPDH mRNA4: Y = 15.487+0.936X R~2=0.965, slope rate was 0.936GAPDH mRNA5: Y = 15.580 + 0.892X R~2=0.949, slope rate was 0.892GAPDH mRNA6: Y = 15.570 + 0.829X R~2=0.956, slope rate was 0.829The slope rate of GAPDH mRNA 4-6 degradation were lower than GAPDH mRNA 1-3. If we used GAPDH mRNA 6 as external standard, the ratio of GAPDH mRNA 1-3/GAPDH mRNA 6 were correlated with postmortem periods. The regression equations are as follows:GAPDH mRNA 1/GAPDH mRNA 6: Y = 1.015+0.029 X , R~2=0.898GAPDH mRNA 2/GAPDH mRNA 6 :Y = 1.028+0.030X , R~2=0.871GAPDH mRNA3/GAPDH mRNA6:Y = 1.031+0.028X , R~2=0.879However, the relation between GAPDH 4-5/GAPDH 6 and postmortem periods were not observed.Conclusions (1) The extraction of total RNA was the most key step in researching for mRNA degradation in forensic experiments. The two different commercialize reagents are basically satisfied with RT-PCR. However, from the point of view in quality control and comparability of results, SV Total RNA Isolation System is better for forensic experiments. (2) The SYBR Green I real-time RT-PCR was a reliable technique for researching mRNA degradation compare to one step RT-PCR technique. Adoption of the housekeeping genes for using to research mRNA degradation eliminated systematical errors of other genes. As the objective index, C_t values have a good correlation with postmortem periods, It was a ideal index for estimating postmortem intervals especially in advanced stage PMI. (3) The results of tissue variability researching showed that mRNAs of brain and spleen in rats were more stable than that from other organs especially in advanced stage PMI although ambient temperature and humidity should be considered as the important factors in estimation of death time. (4) The different sites of the same housekeeping gene in the same tissue share the different degradation rates, The reason might be the degradation started from the 5' -mRNA cap structure to the 3' -end in the condition of RNA integrality. Thus, selection of the primer pairs should be near to the 3' -end of mRNA. The sequence s of primers should be identical so as to easily compare to results among different forensic labs.(5) We provide a novel approach and new view to estimate death time by mRNA real time RT-PCR analysis in forensic casework.