大鼠死后看家基因mRNA降解规律与晚期死亡时间推断的相关性研究
发布时间：2018-09-01 14:30
【摘要】： 目的：本研究从基因库中筛选出两种稳定表达于不同个体与组织细胞中的看家基因GAPDH和β-actin，采用实时荧光定量RT-PCR技术研究大鼠死后不同时间和不同脏器中两种看家基因的mRNA降解规律，建立mRNA降解的程度与死亡时间关系的回归方程，探索用于晚期死亡时间推断较为理想的组织，并试图建立标准化的RNA提取与定量检测方法，，希望能最终应用于法医学实践中。 方法：(1)选取SD大鼠28只，按不同死后经过时间分对照组(死后即刻)和实验组1～6(死后1天、3天、5天、7天、9天、12天)，每组4只，对照组脊椎脱臼法处死后快速取出心、肝、脾、肺、肾、脑，称量多个小块(1g)组织置于液氮中保存备用。实验组脊椎脱臼法处死后置于人工气候箱内，设定条件为温度20℃，湿度50％，白天、黑夜各12小时。分别于1天、3天、5天、7天、9天、12天后各取出4只大鼠的心、肝、脾、肺、肾、脑，称量多个小块(1g)组织置于液氮中保存备用。(2)采用以膜提取技术(不含苯酚)为基础和以异硫氰酸胍／苯酚法为原理的两种商品化试剂盒对大鼠心、肝、脾、肺、肾、脑组织进行总RNA的提取，并进行总RNA的评价与鉴定，以分析评价两种提取方法的优劣及适用范围。(3)对组织中GAPDH mRNA和β-actin mRNA进行一步法RT-PCR检测，应用凝胶图像分析系统分别对各组织中GAPDH mRNA和β-actin mRNA扩增产物电泳图目标条带进行灰度扫描和光密度分析，计算出各条带相对灰度值和光密度值，结果以相对灰度(Relative grayscale)和积分光密度值(Integral optical density，IOD)表示，并筛选出在死后较长时间段仍能检出的组织脏器。(4)根据一步法RT-PCR扩增后产物凝胶电泳的实验结果，本文选取了在死后5天内仍能检测出扩增产物的脑组织和脾脏，使用SYBR GreenⅠ嵌合荧光法实时荧光定量RT-PCR技术，根据荧光信号的累积情况实时监测GAPDH mRNA和β-actin mRNA不同时间点的RT-PCR进程，并将PMI延长至12天，结果用循环阈值(简称C_t值)表示，分析死后经过时间与C_t值的线性关系，并最终建立死亡时间推断回归方程。(5)为探讨同一种看家基因上的不同位点，其降解速率是否也有一定规律性，本文选取了在死后较长时间内仍能检测出扩增产物的脑组织，对GAPDH mRNA的6个不同位点进行不同时间点的实时荧光定量RT-PCR监测，这些位点依次分布于GAPDH mRNA的5'～3'端。并将PMI时间延长、间隔增加(死后即刻、1天、5天、9天、12天)，结果用循环阈值(简称C_t值)表示。通过检测大鼠死后不同时间脑组织中各位点GAPDH mRNA的降解，分析不同位点的降解规律及其与死后经过时间的相关性，试图寻找单一看家基因的降解规律。 结果：(1)总RNA评价与鉴定均以对照组为例作比较，采用TRIzol试剂盒与SVTotal RNA Isolation System提取的总RNA纯度与得率均可满足下一步实验要求。各组织OD_(260／280)均在1．9～2．1之间，结果无显著性差异(P0．01)。但在SV Total RNA Isolation System中加入了DNaseⅠ消化，有效的去除了DNA污染，因此提取成功率明显高于TRIzol试剂盒。除SV Total RNA Isolation System对组织提取上限量要求较低外，不同提取方法获得的各组织脏器总RNA得率无显著性差异。各组织脏器中总RNA得率依次为肝脏脾脏心脏大脑肾脏肺脏。均可满足下一步实验要求。(2)GAPDH mRNA、β-actin mRNA进行RT-PCR反应后，扩增产物的相对灰度与积分光密度值随死后经过时间的延长而逐渐减小，且与死亡时间显著相关，从扩增产物电泳图中可见大鼠脾脏和脑组织GAPDH mRNA和β-actin mRNA在死后5天内可被检出，心脏和肾脏在死后3天内可被检出，而肝脏和肺脏GAPDH mRNA和β-actin mRNA降解较快，仅在死后1天内被检出。以脾脏和脑组织检出时间最长。(3)使用SYBR GreenⅠ嵌合法实时荧光定量RT-PCR，根据荧光信号的累积实时监测脑组织和脾脏GAPDH mRNA和β-actin mRNA不同时间点的RT-PCR进程，经线性回归分析，GAPDH mRNA和β-actin mRNA的C_t值均与PMI之间存在显著的相关性，并得出死亡时间推断的回归方程(脑组织GAPDH：Y=15．312+1．531X，R~2=0．943；脑组织β-actin：Y=15．609+1．750X，R~2=0．953；脾脏GAPDH：Y=19．571+1．453X，R~2=0．852；脾脏β-actin：Y=21．769+1．274X，R~2=0．808)。(4)经线性回归分析，脑组织中各位点GAPDH mRNA均与死后经过时间有相关性，但不同位点的mRNA降解速率不同。各位点线性回归方程如下：GAPDH mRNA1：Y=15．501+1．577X R~2=0．968，斜率为1．577GAPDH mRNA2：Y=15．717+1．596X R~2=0．980，斜率为1．596GAPDH mRNA3：Y=15．772+1．553X R~2=0．973，斜率为1．553GAPDH mRNA4：Y=15．487+0．936X R~2=0．965，斜率为0．936GAPDH mRNA5：Y=15．580+0．892X R~2=0．949，斜率为0．892GAPDH mRNA6：Y=15．570+0．829X R~2=0．956，斜率为0．829各个线性回归方程中，GAPDH mRNA1～GAPDH mRNA3的斜率为1．577、1．596、1．553，这三个位点的降解速率基本相同。GAPDH mRNA4～GAPDH mRNA6的斜率为0．936、0．892、0．829，这三个位点的降解速率基本相同。但与前三个位点相比，斜率减小，说明后三个位点mRNA的降解速率要慢于前三个位点。以GAPDH mRNA 6作为外标，GAPDHmRNA1～GAPDH mRNA3与其的比值同PMI之间存在显著的相关性(回归方程分别为Y=1．015+0．029X，R~2=0．898；Y=1．028+0．030X，R~2=0．871；Y=1．031+0．028X，R~2=0．879)。而GAPDH mRNA4和GAPDH mRNA 5与作为外标的GAPDH mRNA 6的比值同PMI之间无相关性。 结论：(1)在研究机体死后mRNA降解规律的实验中，总RNA的提取是实验成功与否的关键，而不同商品化提取试剂均可满足实验要求，但从法医学实验室质量控制及检验结果是否具有可比性的角度出发，SV Total RNA Isolation System效果更好。(2)与一步法RT-PCR技术相比，SYBR GreenⅠ嵌合法实时荧光定量RT-PCR在定量分析mRNA降解的研究中是一个更理想的技术手段。选用看家基因作为PMI推断的研究对象，可在法医检案中消除其它基因因为个体差异带来的误差，更具实用性。C_t值作为动态监测机体死后不同时间点的客观指标，与死后不同时间点的线性关系良好，推断死后经过时间尤其是晚期死亡时间较为理想，(3)死后mRNA特别是看家基因mRNA的组织差异性研究表明，脑组织和脾脏中mRNA稳定性较好，适用于PMI特别是晚期PMI的推断。除环境温度外，环境湿度也是死亡时间推断研究中的重要影响因素，在今后的研究中应加以考虑。(4)同一种组织中，同一种看家基因的不同位点存在降解速率的差异性。造成这种差异性的原因可能是机体死亡后，在保证RNA分子完整性的前提下，mRNA的降解可能是从5'端向3'端进行的。因此选取看家基因作为引物时，应尽量选取靠近3'端的位点，这些位点降解速率慢，可能更适于研究晚期死亡时间的推断。同时在实验室结果比较中，也应将引物序列标准化，便于不同实验室的结果比较与评价。(5)采用RNA尤其是mRNA作为研究目标进行死亡时间推断为此研究领域提供了一个新的思路。
[Abstract]:AIM: To screen two kinds of housekeeping genes GAPDH and beta-actin stably expressed in different individuals and tissues and cells from the gene pool. Real-time fluorescence quantitative RT-PCR was used to study the mRNA degradation of two kinds of housekeeping genes in different organs and different time after death in rats, and to establish the relationship between the degree of mRNA degradation and the time of death. Regression equation is used to explore the ideal tissue for estimating the time of late death, and to establish a standardized method for RNA extraction and quantitative detection, hoping to be eventually applied in forensic practice.
Methods: (1) Twenty-eight SD rats were divided into control group (immediately after death) and experimental group (1-6 days, 3 days, 5 days, 7 days, 9 days, 12 days) according to different postmortem passage time. Four rats in each group were selected. In control group, the heart, liver, spleen, lung, kidney, brain were quickly removed after vertebral dislocation. After death, the rats were placed in an artificial climate chamber under the following conditions: temperature 20 C, humidity 50%, day and night 12 hours. The hearts, livers, spleens, lungs, kidneys, and brains of four rats were taken out after 1 day, 3 days, 5 days, 7 days, 9 days, and 12 days respectively, and several small pieces (1g) of tissue were weighed and stored in liquid nitrogen. Two commercial kits based on guanidine isothiocyanate/phenol method were used to extract total RNA from heart, liver, spleen, lung, kidney and brain of rats, and the total RNA was evaluated and identified in order to analyze the advantages and disadvantages of the two methods and evaluate their applicability. (3) GAPDH mRNA and beta-actin mRNA in tissues were detected by one-step RT-PCR, and gel was used. The image analysis system scans the target bands of GAPDH mRNA and beta-actin mRNA amplification products electrophoresis map in each tissue by gray scale scanning and optical density analysis, and calculates the relative gray value and optical density value of each band. The results are expressed by relative gray scale (Relative gray scale) and integral optical density (IOD). (4) According to the results of one-step RT-PCR amplified product gel electrophoresis, brain tissues and spleens which could detect the amplified products within 5 days after death were selected. The real-time fluorescence quantitative RT-PCR technique of SYBR Green I chimeric fluorescence was used to detect the amplified products according to the accumulation of fluorescent signals. The RT-PCR process of GAPDH mRNA and beta-actin mRNA at different time points was monitored and the PMI was prolonged to 12 days. Results The linear relationship between postmortem passage time and C_t value was analyzed by cyclic threshold value, and the regression equation was established to deduce the postmortem time. (5) To explore the different sites in the same housekeeping gene, the degradation rate was as follows. In this study, six different sites of GAPDH mRNA were monitored by real-time fluorescence quantitative RT-PCR at different time points. These sites were distributed in the 5'-3'end of GAPDH mRNA in turn. The PMI time was prolonged and the interval was increased (immediately after death, 1 day, 5 days). By detecting the degradation of GAPDH mRNA at different sites in brain tissues at different time points after death, we analyzed the degradation regularity of different sites and their correlation with postmortem passage time, and tried to find the degradation regularity of single housekeeper gene.
Results: (1) Total RNA evaluation and identification were compared with the control group. The purity and yield of total RNA extracted by TRIzol kit and SVTotal RNA Isolation System could meet the next experimental requirements. OD_ (260/280) of all tissues ranged from 1.9 to 2.1, and the results showed no significant difference (P 0.01). DNA contamination was effectively removed by adding DNase I digestion, so the success rate of extraction was significantly higher than that of TRIzol kit. Except for SV Total RNA Isolation System, which requires a lower upper limit of tissue extraction, there was no significant difference in the total RNA yield of different tissues obtained by different methods. The relative gray and integral optical density of the amplified products decreased with the prolongation of postmortem time, and were significantly correlated with the time of death. GAPDH in the spleen and brain tissues of rats could be seen from the electrophoresis of the amplified products. MRNA and beta-actin mRNA could be detected within 5 days after death, heart and kidney could be detected within 3 days after death, while GAPDH mRNA and beta-actin mRNA in liver and lung degraded more rapidly, only detected within 1 day after death. Spleen and brain tissues were detected for the longest time. (3) SYBR Green I embedded real-time fluorescent quantitative RT-PCR was used according to the accumulation of fluorescent signals. Real-time RT-PCR of GAPDH mRNA and beta-actin mRNA in brain and spleen was monitored. Linear regression analysis showed that there was a significant correlation between C_t values of GAPDH mRNA and beta-actin mRNA and PMI. The regression equation for estimating the time of death (GAPDH: Y = 15.312 + 1.531X, R~2 = 0.943; beta-actin in brain tissue) was obtained. Y = 15.609 + 1.750X, R~2 = 0.953; spleen GAPDH: Y = 19.571 + 1.453X, R~2 = 0.852; spleen beta-actin: Y = 21.769 + 1.274X, R~2 = 0.808). (4) By linear regression analysis, the GAPDH mRNA in brain tissues were correlated with postmortem time, but the mRNA degradation rates at different sites were different. GAPDH mRNA1:Y = 15.501+15.501+1.577X R~2 = 0.968, with a slope of 1.577 GAPDH mRNA2:Y = 15.717 + 1.596X R~2 = 0.980, with a slope of 1.596 GAPDH mRNA3:Y = 15.772 + 15.772 + 1.772 + 1.553X R~2 = 0.973, and 1.553GGAPDH mRNA4:Y = 4:Y = 15.487 + 0.936X R~2 = 15.487 + 0.936X R~2 = 0.965, and 1.577 GAPDH mRNA4:Y = 15.487 + 0.487 + 0.936X R~2 = 0.965, and 0.2X R~2=0.949 The slope of GAPDH mRNA 6:Y=15.570+0.829XR~2=0.956, and the slope of GAPDH mRNA 1-GAPDH mRNA 3 was 1.577,1.596,1.553 in each linear regression equation of 0.829. The degradation rates of the three sites were basically the same. The slope of GAPDH mRNA 4-GAPDH mRNA 6 was 0.936,0.892,0.829, and the degradation rates of the three sites were 1.577,1.596,1.553. Compared with the first three sites, the slope decreased, indicating that the degradation rate of mRNA in the latter three sites was slower than that in the former three sites. R~2=0.871; Y=1.031+0.028X, R~2=0.879). However, the ratio of GAPDH mRNA 4 and GAPDH mRNA 5 to GAPDH mRNA 6 as an external standard had no correlation with PMI.
Conclusion: (1) The extraction of total RNA is the key to the success of the experiment in studying the degradation of postmortem mRNA. Different commercial extracting reagents can meet the experimental requirements, but SV Total RNA Isolation System is more effective from the point of view of quality control and comparability of test results in forensic laboratory. Compared with one-step RT-PCR, SYBR Green I real-time fluorescence quantitative RT-PCR is a more ideal technique for quantitative analysis of mRNA degradation. Choosing the housekeeping gene as the research object of PMI inference can eliminate the errors caused by individual differences in forensic medical records and is more practical. The objective indexes of postmortem monitoring at different time points have a good linear relationship with postmortem time points, and the postmortem passage time, especially the late postmortem time, is more ideal. (3) The study on the tissue differences of postmortem mRNA, especially the home-care gene mRNA, shows that the mRNA in brain tissue and spleen is stable and suitable for PMI, especially in the late stage. PMI inference. In addition to ambient temperature, ambient humidity is also an important factor in the study of time-to-death inference, which should be taken into account in future studies. (4) In the same tissue, there are differences in degradation rates at different sites of the same housekeeping gene. The reason for this difference may be that RNA molecules are guaranteed after death. In the premise of integrity, the degradation of mRNA may occur from the 5'end to the 3'end. Therefore, when selecting the watcher gene as a primer, we should try to select sites close to the 3'end. The degradation rate of these sites is slow, which may be more suitable for studying the inference of late-stage mortality. Comparing and evaluating the results with the laboratory. (5) Using RNA, especially mRNA, as the research target to infer the time of death provides a new idea for this research field.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：D919;Q953
本文编号：2217492
[Abstract]:AIM: To screen two kinds of housekeeping genes GAPDH and beta-actin stably expressed in different individuals and tissues and cells from the gene pool. Real-time fluorescence quantitative RT-PCR was used to study the mRNA degradation of two kinds of housekeeping genes in different organs and different time after death in rats, and to establish the relationship between the degree of mRNA degradation and the time of death. Regression equation is used to explore the ideal tissue for estimating the time of late death, and to establish a standardized method for RNA extraction and quantitative detection, hoping to be eventually applied in forensic practice.
Methods: (1) Twenty-eight SD rats were divided into control group (immediately after death) and experimental group (1-6 days, 3 days, 5 days, 7 days, 9 days, 12 days) according to different postmortem passage time. Four rats in each group were selected. In control group, the heart, liver, spleen, lung, kidney, brain were quickly removed after vertebral dislocation. After death, the rats were placed in an artificial climate chamber under the following conditions: temperature 20 C, humidity 50%, day and night 12 hours. The hearts, livers, spleens, lungs, kidneys, and brains of four rats were taken out after 1 day, 3 days, 5 days, 7 days, 9 days, and 12 days respectively, and several small pieces (1g) of tissue were weighed and stored in liquid nitrogen. Two commercial kits based on guanidine isothiocyanate/phenol method were used to extract total RNA from heart, liver, spleen, lung, kidney and brain of rats, and the total RNA was evaluated and identified in order to analyze the advantages and disadvantages of the two methods and evaluate their applicability. (3) GAPDH mRNA and beta-actin mRNA in tissues were detected by one-step RT-PCR, and gel was used. The image analysis system scans the target bands of GAPDH mRNA and beta-actin mRNA amplification products electrophoresis map in each tissue by gray scale scanning and optical density analysis, and calculates the relative gray value and optical density value of each band. The results are expressed by relative gray scale (Relative gray scale) and integral optical density (IOD). (4) According to the results of one-step RT-PCR amplified product gel electrophoresis, brain tissues and spleens which could detect the amplified products within 5 days after death were selected. The real-time fluorescence quantitative RT-PCR technique of SYBR Green I chimeric fluorescence was used to detect the amplified products according to the accumulation of fluorescent signals. The RT-PCR process of GAPDH mRNA and beta-actin mRNA at different time points was monitored and the PMI was prolonged to 12 days. Results The linear relationship between postmortem passage time and C_t value was analyzed by cyclic threshold value, and the regression equation was established to deduce the postmortem time. (5) To explore the different sites in the same housekeeping gene, the degradation rate was as follows. In this study, six different sites of GAPDH mRNA were monitored by real-time fluorescence quantitative RT-PCR at different time points. These sites were distributed in the 5'-3'end of GAPDH mRNA in turn. The PMI time was prolonged and the interval was increased (immediately after death, 1 day, 5 days). By detecting the degradation of GAPDH mRNA at different sites in brain tissues at different time points after death, we analyzed the degradation regularity of different sites and their correlation with postmortem passage time, and tried to find the degradation regularity of single housekeeper gene.
Results: (1) Total RNA evaluation and identification were compared with the control group. The purity and yield of total RNA extracted by TRIzol kit and SVTotal RNA Isolation System could meet the next experimental requirements. OD_ (260/280) of all tissues ranged from 1.9 to 2.1, and the results showed no significant difference (P 0.01). DNA contamination was effectively removed by adding DNase I digestion, so the success rate of extraction was significantly higher than that of TRIzol kit. Except for SV Total RNA Isolation System, which requires a lower upper limit of tissue extraction, there was no significant difference in the total RNA yield of different tissues obtained by different methods. The relative gray and integral optical density of the amplified products decreased with the prolongation of postmortem time, and were significantly correlated with the time of death. GAPDH in the spleen and brain tissues of rats could be seen from the electrophoresis of the amplified products. MRNA and beta-actin mRNA could be detected within 5 days after death, heart and kidney could be detected within 3 days after death, while GAPDH mRNA and beta-actin mRNA in liver and lung degraded more rapidly, only detected within 1 day after death. Spleen and brain tissues were detected for the longest time. (3) SYBR Green I embedded real-time fluorescent quantitative RT-PCR was used according to the accumulation of fluorescent signals. Real-time RT-PCR of GAPDH mRNA and beta-actin mRNA in brain and spleen was monitored. Linear regression analysis showed that there was a significant correlation between C_t values of GAPDH mRNA and beta-actin mRNA and PMI. The regression equation for estimating the time of death (GAPDH: Y = 15.312 + 1.531X, R~2 = 0.943; beta-actin in brain tissue) was obtained. Y = 15.609 + 1.750X, R~2 = 0.953; spleen GAPDH: Y = 19.571 + 1.453X, R~2 = 0.852; spleen beta-actin: Y = 21.769 + 1.274X, R~2 = 0.808). (4) By linear regression analysis, the GAPDH mRNA in brain tissues were correlated with postmortem time, but the mRNA degradation rates at different sites were different. GAPDH mRNA1:Y = 15.501+15.501+1.577X R~2 = 0.968, with a slope of 1.577 GAPDH mRNA2:Y = 15.717 + 1.596X R~2 = 0.980, with a slope of 1.596 GAPDH mRNA3:Y = 15.772 + 15.772 + 1.772 + 1.553X R~2 = 0.973, and 1.553GGAPDH mRNA4:Y = 4:Y = 15.487 + 0.936X R~2 = 15.487 + 0.936X R~2 = 0.965, and 1.577 GAPDH mRNA4:Y = 15.487 + 0.487 + 0.936X R~2 = 0.965, and 0.2X R~2=0.949 The slope of GAPDH mRNA 6:Y=15.570+0.829XR~2=0.956, and the slope of GAPDH mRNA 1-GAPDH mRNA 3 was 1.577,1.596,1.553 in each linear regression equation of 0.829. The degradation rates of the three sites were basically the same. The slope of GAPDH mRNA 4-GAPDH mRNA 6 was 0.936,0.892,0.829, and the degradation rates of the three sites were 1.577,1.596,1.553. Compared with the first three sites, the slope decreased, indicating that the degradation rate of mRNA in the latter three sites was slower than that in the former three sites. R~2=0.871; Y=1.031+0.028X, R~2=0.879). However, the ratio of GAPDH mRNA 4 and GAPDH mRNA 5 to GAPDH mRNA 6 as an external standard had no correlation with PMI.
Conclusion: (1) The extraction of total RNA is the key to the success of the experiment in studying the degradation of postmortem mRNA. Different commercial extracting reagents can meet the experimental requirements, but SV Total RNA Isolation System is more effective from the point of view of quality control and comparability of test results in forensic laboratory. Compared with one-step RT-PCR, SYBR Green I real-time fluorescence quantitative RT-PCR is a more ideal technique for quantitative analysis of mRNA degradation. Choosing the housekeeping gene as the research object of PMI inference can eliminate the errors caused by individual differences in forensic medical records and is more practical. The objective indexes of postmortem monitoring at different time points have a good linear relationship with postmortem time points, and the postmortem passage time, especially the late postmortem time, is more ideal. (3) The study on the tissue differences of postmortem mRNA, especially the home-care gene mRNA, shows that the mRNA in brain tissue and spleen is stable and suitable for PMI, especially in the late stage. PMI inference. In addition to ambient temperature, ambient humidity is also an important factor in the study of time-to-death inference, which should be taken into account in future studies. (4) In the same tissue, there are differences in degradation rates at different sites of the same housekeeping gene. The reason for this difference may be that RNA molecules are guaranteed after death. In the premise of integrity, the degradation of mRNA may occur from the 5'end to the 3'end. Therefore, when selecting the watcher gene as a primer, we should try to select sites close to the 3'end. The degradation rate of these sites is slow, which may be more suitable for studying the inference of late-stage mortality. Comparing and evaluating the results with the laboratory. (5) Using RNA, especially mRNA, as the research target to infer the time of death provides a new idea for this research field.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：D919;Q953
【引证文献】
相关硕士学位论文 前1条
1 王颖希;RNA用于体液鉴别的方法学建立及其法医学应用研究[D];山西医科大学;2012年
本文编号：2217492
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