Combined metabolomics and machine learning algorithms to explore metabolic biomarkers for diagnosis of acute myocardial ischemia.

Acute myocardial ischemia (AMI) remains the leading cause of death worldwide, and the post-mortem diagnosis of AMI represents a current challenge for both clinical and forensic pathologists. In the present study, the untargeted metabolomics based on ultra-performance liquid chromatography combined with high-resolution mass spectrometry was applied to analyze serum metabolic signatures from AMI in a rat model (n = 10 per group). A total of 28 endogenous metabolites in serum were significantly altered in AMI group relative to control and sham groups. A set of machine learning algorithms, namely gradient tree boosting (GTB), support vector machine (SVM), random forest (RF), logistic regression (LR), and multilayer perceptron (MLP) models, was used to screen the more valuable metabolites from 28 metabolites to optimize the biomarker panel. The results showed that classification accuracy and performance of MLP model were better than other algorithms when the metabolites consisting of L-threonic acid, N-acetyl-L-cysteine, CMPF, glycocholic acid, L-tyrosine, cholic acid, and glycoursodeoxycholic acid. Finally, 17 blood samples from autopsy cases were applied to validate the classification model's value in human samples. The MLP model constructed based on rat dataset achieved accuracy of 88.23%, and ROC of 0.89 for predicting AMI type II in autopsy cases of sudden cardiac death. The results demonstrated that MLP model based on 7 molecular biomarkers had a good diagnostic performance for both AMI rats and autopsy-based blood samples. Thus, the combination of metabolomics and machine learning algorithms provides a novel strategy for AMI diagnosis.

A novel method for determining postmortem interval based on the metabolomics of multiple organs combined with ensemble learning techniques.

Determining postmortem interval (PMI) is one of the most challenging and essential endeavors in forensic science. Developments in PMI estimation can take advantage of machine learning techniques. Currently, applying an algorithm to obtain information on multiple organs and conducting joint analysis to accurately estimate PMI are still in the early stages. This study aimed to establish a multi-organ stacking model that estimates PMI by analyzing differential compounds of four organs in rats. In a total of 140 rats, skeletal muscle, liver, lung, and kidney tissue samples were collected at each time point after death. Ultra-performance liquid chromatography coupled with high-resolution mass spectrometry was used to determine the compound profiles of the samples. The original data were preprocessed using multivariate statistical analysis to determine discriminant compounds. In addition, three interrelated and increasingly complex patterns (single organ optimal model, single organ stacking model, multi-organ stacking model) were established to estimate PMI. The accuracy and generalized area under the receiver operating characteristic curve of the multi-organ stacking model were the highest at 93% and 0.96, respectively. Only 1 of the 14 external validation samples was misclassified by the multi-organ stacking model. The results demonstrate that the application of the multi-organ combination to the stacking algorithm is a potential forensic tool for the accurate estimation of PMI.

Exploration and Practice of the "One Combination, Two Highlights, Three Combinations, Four in One" Innovative Talents Training Mode in Forensic Medicine.

Talent is one of the basic and strategic supports for building a modern socialist country in all aspects. Since the 1980s, the establishment of forensic medicine major and the cultivation of innovative talents in forensic medicine have become hot topics in higher education in forensic medicine. Over the past 43 years, the forensic medicine team of Shanxi Medical University has adhered to the joint education of public security and colleges, and made collaborative innovation, forming a training mode of "One Combination, Two Highlights, Three Combinations, Four in One" for innovative talents in forensic medicine. It has carried out "5+3/X" integrated reform, and formed a relatively complete talent training innovation mode and management system in teaching, scientific research, identification, major, discipline, team, platform and cultural construction. It has made a historic contribution to China's higher forensic education, accumulated valuable experience for the construction of first-class major and first-class discipline of forensic medicine, and provided strong support for the construction of the national new forensic talent training system. The popularization of this training mode is conducive to the rapid and sustainable development of forensic science, and provides more excellent forensic talents for national building, regional social development and the discipline construction of forensic science.

Pharmacokinetics of meperidine (pethidine) in rabbit oral fluid: correlation with plasma concentrations after controlled administration.

Oral fluid assays for quantifying drugs are useful in forensic toxicology and drug monitoring. Compared with blood and urine specimens, oral fluid collection is simple, non-invasive, and more difficult to adulterate. Therefore, we investigated whether meperidine and its metabolites could be detected in oral fluid and whether there was a predictable relationship between oral fluid and plasma concentrations. Male New Zealand white rabbits (n = 10) were administered meperidine hydrochloride (20 mg/kg, intravenous). Then, plasma and oral fluid were collected at various time points up to 10 h after administration. We developed a simple and sensitive gas chromatography-mass spectrometry method for the determination of meperidine and normeperidine in oral fluid and plasma. We estimated the apparent pharmacokinetic parameters for meperidine in oral fluid and plasma and determined the ratio and correlation between oral fluid and plasma concentrations. The results demonstrate that this method has excellent specificity, linearity, precision, and recovery. Meperidine and normeperidine were detected in both body fluids; meperidine was the most abundant analyte in oral fluid. The oral fluid-to-plasma drug concentration ratios did not differ significantly over time (p > 0.05). In addition, oral fluid and plasma levels of meperidine and normeperidine were significantly correlated over time (r = 0.713 and 0.725, respectively; p < 0.05). These results provide context for interpreting meperidine and metabolite concentrations in oral fluid and support the utility of oral fluid as an alternative matrix in clinical and forensic testing.

[Relation between Injury Time and the Expression of COX6C mRNA in Skeletal Muscle of Rats after Contusion].

OBJECTIVE: To investigate the relation between injury time and the expression of cytochrome c oxidase subunit VIc (COX6C) mRNA in skeletal muscle of rat after contusion. METHODS: A total of fifty-four SD rats were divided into the control group and the contusion groups (0.5, 1, 6, 12, 18, 24, 30, and 36 h after contusion), randomly. The contusion model was established by free fall drop of gravity hammer. At corresponding time point after contusion, the regular histology was examined and expression level of COX6C mRNA was tested by real-time PCR after extraction of total RNA from the tissues. RESULTS: The main pathological features of 6 h after injury included edema and hemorrhage in myocytes with no inflammatory cells found. After 6 hours, the findings included myocyte degeneration and necrosis, inflammatory cells infiltration, and fibrous connective tissue proliferation in the contused zone. The expression level of COX6C mRNA was higher than that of the control group within 6 h after contusion. The expression level was lower than that of the control group from 6-36 h after contusion. CONCLUSION: The level of COX6C mRNA expresses in a regular way after contusion. It may be useful for estimating wound age in combination with the results of pathological features.

Relative Expression of Indicators for Wound Age Estimation in Forensic Pathology.

OBJECTIVE: In order to understand which kind of function genes play an important role for estimating wound age, the variation of difference genes' mRNA expression were compared after injury. METHODS: The mRNA expression levels of seven candidate genes (ICAM-1, NF-κB, MX2, MT1, MT2, sTnI, and Cox6c) were analyzed in contused rat skeletal muscle at different time points using real-time fluorescent quantitative PCR (RT-qPCR). The raw Ct values were normalized relative to that of RPL32 mRNA, and converted to standard Ct values. At each time point after injury, the standard deviations (SD) of the standard Ct values were calculated by SPSS. RESULTS: The expression trends of the seven genes were all found to be related to wound age, but there were lower variation coefficients and greater reliability of s TnI and Cox6c when compared with other genes. CONCLUSION: The genes encoding structural proteins or proteins that perform basic functions can be suitable for wound age estimation.

[Expression of SFRP5 mRNA in Rat Skeletal Muscle after Contusion].

OBJECTIVE: To investigate the relationship between the expression of secreted frizzled-related protein 5 (SFRP5) mRNA and the time interval after skeletal muscle injury in rats by real-time PCR. METHODS: A total of ninety SD rats were randomly divided into the contusion groups at different times including 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h after contusion, incision groups at different times including 4h and 8h after incision and the control group. The samples were taken from the contused zone at different time points. The total RNA was isolated from the samples and reversely transcribed to analyze the expression levels of SFRP5 mRNA. RESULTS: Compared to the control group, the expression of SFRP5 mRNA in contusion groups were down-regulated within 48 h after contusion and reached the lowest level at 20 h, and the expression of SFRP5 mRNA gradually increased from 20 h to 48 h after contusion. The expression of SFRP5 mRNA in the incised groups were significantly lower than that of the contusion groups at 4 h after injury. At the time of 8 h, the expression levels between the contusion and incision groups showed no statistically significant difference. CONCLUSION: It is suggested that SFRP5 mRNA analysis may show regular expression and can be a marker for estimation of skeletal muscle injury age.

[Protective effects of intermedin preconditioning on hypoxic injury in rat's cardiac myocytes].

OBJECTIVE: To observe the effects of intermedin preconditioning on hypoxic injury in rat's cardiac myocytes and to provide the hypothetical mechanism of sudden cardiac death in the field of forensic pathology. METHODS: The H9c2 cultured rat cardiac myocytes were randomly divided into control group, hypoxia group and IMD group. The myocardial cell viability, cellular ultrastructure, intracellular calcium concentration and apoptosis rate were determined by MTT assay, transmission electron microscopy, laser scanning confocal microscope and flow cytometry, respectively. RESULTS: Compared with the control group, cell viability obviously decreased with inner ultrastructure injury in the hypoxia group (P<0.05), while cell viability significantly increased in the IMD group by reducing the hypoxia injury of cardiac myocytes (P<0.05). Compared with the control group, [Ca2+]i (fluorescence intensity) and apoptosis rate significantly increased in the hypoxia group, but decreased in the IMD group (P<0.05). CONCLUSION: IMD increases the cell survival rate and decreases the cell apoptosis inhibited by intracellular calcium overload from hypoxia. This finding may reveal the mechanism of protective effects of myocardial hypoxia, and provide a scientific basis for the identification sudden cardiac death.

Time-dependent expression of SNAT2 mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation.

To estimate the age of skeletal muscle contusion, the expression of SNAT2 mRNA in contused skeletal muscle of rats was detected by real-time polymerase chain reaction (PCR). In total, 78 Sprague-Dawley male rats were divided into control and contusion groups. At 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h (n = 6) after contusion, the rats were sacrificed with a lethal dose of pentobarbital. Another 24 rats received contusion injuries at 6, 12, 18, and 24 h (n = 6) after death. Total RNA was isolated from muscle specimens using the TRIzol reagent and reverse-transcribed into first-strand cDNA. Sequence-specific primers and TaqMan fluorogenic probes for SNAT2 mRNA and RPL13 mRNA were designed using the AlleleID 6 software, and the expression levels of SNAT2 mRNA were determined by real-time PCR. At 4, 16, 20, and 24 h after contusion, expression levels of SNAT2 mRNA normalized to RPL13 mRNA increased by 2.07 (P < 0.05), 2.53 (P < 0.05), 2.68 (P < 0.05), and 2.06 fold (P < 0.05) respectively, versus that in the control group. However, there was no significant change in the expression level of SNAT2 mRNA from 24 to 48 h (P > 0.05) after contusion, when normalized to RPL13 mRNA. There was no change in the expression level of SNAT2 mRNA between the normal skeletal muscle from the left limb of the same injured rat and the control. Also, no degradation of SNAT2 mRNA was detected in the postmortem samples (P > 0.05). This result suggests that the determination of SNAT2 mRNA levels by real-time PCR may be useful for estimating wound age.

Cost-effectiveness analysis of pembrolizumab plus chemotherapy versus chemotherapy as the first-line treatment for advanced esophageal cancer.

OBJECTIVE: The KEYNOTE-590 trial showed that individuals with advanced esophageal cancer who received Pembrolizumab in combination with chemotherapy as a first-line regimen achieved a significant extension of survival. However, this treatment option increases the financial burden on patients and the economic benefits remain to be further evaluated. METHODS: A Markov model was used to simulate 10-year survival of patients with esophageal cancer from the perspective of United States (US) Medicare payers. We evaluated the economics of Pembrolizumab plus chemotherapy in the PD-L1 positive score (CPS ≥10) and any PD-L1 expression groups, respectively. We estimated total costs, quality-adjusted life years (QALYs), and calculated incremental cost effectiveness ratios (ICERs). Sensitivity analyses were conducted to explore the impact of uncertainties on the results. Subgroup analysis was also performed. RESULTS: The analysis results showed that the ICER for pembrolizumab plus chemotherapy versus chemotherapy alone was $293,513.17/QALYs in the any PD-L1 expression group. This exceeded the threshold of willingness to pay ($150,000/QALYs). ICERs were most sensitive to the cost of pembrolizumab and the ICERs exceeded $150,000/QALYs in all subgroups. CONCLUSIONS: Evidence suggests that first-line pembrolizumab in combination with chemotherapy is not a cost-effective option for advanced esophageal cancer in the US, regardless of PD-L1 expression status.

Exploring postmortem succession of rat intestinal microbiome for PMI based on machine learning algorithms and potential use for humans.

The microbial communities may undergo a meaningful successional change during the progress of decay and decomposition that could aid in determining the post-mortem interval (PMI). However, there are still challenges to applying microbiome-based evidence in law enforcement practice. In this study, we attempted to investigate the principles governing microbial community succession during decomposition of rat and human corpse, and explore their potential use for PMI of human cadavers. A controlled experiment was conducted to characterize temporal changes in microbial communities associated with rat corpses as they decomposed for 30 days. Obvious differences of microbial community structures were observed among different stages of decomposition, especially between decomposition of 0-7d and 9-30d. Thus, a two-layer model for PMI prediction was developed based on the succession of bacteria by combining classification and regression models using machine learning algorithms. Our results achieved 90.48% accuracy for discriminating groups of PMI 0-7d and 9-30d, and yielded a mean absolute error of 0.580d within 7d decomposition and 3.165d within 9-30d decomposition. Furthermore, samples from human cadavers were collected to gain the common succession of microbial community between rats and humans. Based on the 44 shared genera of rats and humans, a two-layer model of PMI was rebuilt to be applied for PMI prediction of human cadavers. Accurate estimates indicated a reproducible succession of gut microbes across rats and humans. Together these results suggest that microbial succession was predictable and can be developed into a forensic tool for estimating PMI.

Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR.

Gene expression profiling by quantitative real-time PCR (RT-qPCR) is a valuable tool in forensic science for estimating the age of a wound. To accurately assess gene expression levels over time in injured tissue, the genes used as internal reference standards must be carefully validated for transcriptional stability. This study examined the transcriptional stability of nine potential reference genes (ß-actin, GAPDH, RPL32, PGK1, SDHA, RPL13, HPRT, Tbp, and Ywhaz) in contused rat skeletal muscle by RT-qPCR. The raw Ct values were determined for each candidate gene at different time points following contusion, and the data were analyzed by the NormFinder, geNorm, and BestKeeper validation programs. The reference genes RPL13 and RPL32 were the most stably expressed genes in contused skeletal muscle, whereas PGK1 was the least stable. The commonly used reference genes ß-actin and GAPDH appeared to be too unstable for normalization of RT-qPCR expression profiling in contused muscle. The reference genes RPL13 and RPL32 were also the best combination for multianalysis. The use of RPL13 and RPL32 as internal standards may improve the accuracy of gene expression studies aimed at determining the age of early wounds in forensic investigations.

[Postmortem pathological changes of actin in skeletal muscles of rats].

OBJECTIVE: To investigate the morphologic changes of actin in skeletal muscles of rats associated with postmortem interval (PMI). METHODS: The morphologic changes of actin filament in the skeletal muscles (adductor magnus of right hind leg) of 28 rats were observed by laser scanning confocal microscope (LSCM) and transmission electron microscope (TEM) at different postmortem intervals (0, 24, 48, 72, 96, 120 and 168 h). RESULTS: The TEM revealed that actin filament began to disintegrate with the lapse of PMI, and eventually the structure of sarcomere and actin filament disappeared. The LSCM showed depletion of anti-actin antibody staining in the skeletal muscles and the extent of staining decreased with extension of PMI correlatively (Y = 0.934-0.005X, R2 = 0.95, P < 0.05). The content of actin positive products reduced with PMI (P < 0.05). No actin positive products were detected 168 h after death. CONCLUSION: The morphologic changes of actin filament in skeletal muscles may become a useful indicator for the estimation of death time.

[Effect and mechanism of intermedin in acute rat cardiac ischemic injury].

OBJECTIVE: To investigate the effect and potential mechanism of intermedin (IMD) in acute cardiac ischemic injury and to provide a new approach for exploring mechanism of sudden cardiac death. METHODS: Seventy-two healthy male rats were randomly divided into 3 groups: control, ischemic and the IMD-treated group. The activity of lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) in heart blood were tested by enzyme chemistry method. The mRNA changes of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) in cardiac were measured by real-time PCR analysis. Myocardial cyclic adenosine monophosphate (cAMP) content was determined by enzyme linked immunosorbent assay (ELISA). Apoptosis related factors Bcl-2 and Bax were detected by immunohistochemistry. RESULTS: Comparing with the control group, LDH and MDA activity of ischemic group in heart blood increased and SOD activity decreased. The concentration of cAMP increased in ventricular muscle, Bcl-2 and Bax proteins expression ratio level decreased. The intravenation of IMD decreased the level of increased activity of LDH and MDA, and lessened the level of decreased activity of SOD. The mRNA expression of CRLR and RAMPs obviously increased in ventricular muscle. CONCLUSION: The protective effect of IMD against myocardial ischemic injury could be caused by decreasing the oxidative stress of ischemia and inhibiting the myocardial apoptosis.

Insight into molecular profile changes after skeletal muscle contusion using microarray and bioinformatics analyses.

Muscle trauma frequently occurs in daily life. However, the molecular mechanisms of muscle healing, which partly depend on the extent of the damage, are not well understood. The present study aimed to investigate gene expression profiles following mild and severe muscle contusion, and to provide more information about the molecular mechanisms underlying the repair process. A total of 33 rats were divided randomly into control (n=3), mild contusion (n=15), and severe contusion (n=15) groups; the contusion groups were further divided into five subgroups (1, 3, 24, 48, and 168 h post-injury; n=3 per subgroup). A total of 2844 and 2298 differentially expressed genes (DEGs) were identified using microarray analyses in the mild and severe contusions, respectively. From the analysis of the 1620 coexpressed genes in mildly and severely contused muscle, we discovered that the gene profiles in functional modules and temporal clusters were similar between the mild and severe contusion groups; moreover, the genes showed time-dependent patterns of expression, which allowed us to identify useful markers of wound age. The functional analyses of genes in the functional modules and temporal clusters were performed, and the hub genes in each module-cluster pair were identified. Interestingly, we found that genes down-regulated at 24-48 h were largely associated with metabolic processes, especially of the oxidative phosphorylation (OXPHOS), which has been rarely reported. These results improve our understanding of the molecular mechanisms underlying muscle repair, and provide a basis for further studies of wound age estimation.

Identifying biomarkers for evaluating wound extent and age in the contused muscle of rats using microarray analysis: a pilot study.

Wound age estimation is still one of the most important and significant challenges in forensic practice. The extent of wound damage greatly affects the accuracy and reliability of wound age estimation, so it is important to find effective biomarkers to help diagnose wound degree and wound age. In the present study, the gene expression profiles of both mild and severe injuries in 33 rats were assayed at 0, 1, 3, 24, 48, and 168 hours using the Affymetrix microarray system to provide biomarkers for the evaluation of wound age and the extent of the wound. After obtaining thousands of differentially expressed genes, a principal component analysis, the least absolute shrinkage and selection operator, and a time-series analysis were used to select the most predictive prognostic genes. Finally, 15 genes were screened for evaluating the extent of wound damage, and the top 60 genes were also screened for wound age estimation in mild and severe injury. Selected indicators showed good diagnostic performance for identifying the extent of the wound and wound age in a Fisher discriminant analysis. A function analysis showed that the candidate genes were mainly related to cell proliferation and the inflammatory response, primarily IL-17 and the Hematopoietic cell lineage signalling pathway. The results revealed that these genes play an essential role in wound-healing and yield helpful and valuable potential biomarkers for further targeted studies.

Time-dependent expression of skeletal muscle troponin I mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation.

To estimate the age of skeletal muscle contusion, the expression of troponin I mRNA in contused skeletal muscle of rats was detected using real-time polymerase chain reaction (PCR). A total of 51 Sprague-Dawley male rats were divided into control and contusion groups, and another nine rats received contusion injury after death. At 0.5, 1, 6, 12, 18, 24, 30, and 36 h after contusion, the rats were killed with a lethal dose of pentobarbital. Total RNA was isolated from muscle specimens using the SV Total RNA Isolation System and reverse transcribed into first-strand cDNA. Sequence-specific primers were then used to conduct real-time PCR to analyze the expression levels of sTnI mRNA. At 0.5, 1, and 6 h after contusion, the expression levels of sTnI mRNA decreased to 78.17% (P < 0.05), 41.58% (P < 0.05), and 32.13% of that in the control group, respectively. However, there were no significant changes in the expression levels of sTnI mRNA from 6 to 36 h (P > 0.05) after contusion when normalized to RpL32 expression. The expression levels of sTnI mRNA in the normal and contused skeletal muscle of postmortem rats were about 70% of that in the control group (P < 0.05), and no significant changes in the expression levels of sTnI mRNA in the postmortem contusion group were noted among different time points after injury. This result suggests that determination of sTnI mRNA levels by real-time PCR is useful for the estimation of wound age.

[Expression of microtubule associated protein-2 mRNA after cerebral contusion].

OBJECTIVE: To explore the mRNA expression of microtubule associated protein-2 (MAP-2) after cerebral contusion, and to investigate its relationship with post-injury interval in a rat model of cerebral contusion. METHODS: Based on Feeney's model of cerebral contusion, the time-dependent changes of MAP-2 mRNA were detected by real-time PCR method in different groups. RESULTS: MAP-2 mRNA showed a lower expression in the brain of the control group. The expression of MAP-2 mRNA decreased 1 h post-injury, and reached the lowest level at 6 h, and the expression of MAP-2 mRNA gradually increased in the focus of the cerebral contusion from 12h to 14d after contusion, which kept a high level up to 14 d post-injury. CONCLUSION: The time-dependent changes of MAP-2 mRNA expression may be a new method for estimating the wound age of cerebral contusion in forensic practice.

[The relationship between GAPDH mRNA multiple location degradation and advanced stage postmortem intervals].

OBJECTIVE: To study the degradation regularity on different sites of housekeeping gene GAPDH in the same tissue in order to deduce advanced stage postmortem interval (PMI). METHODS: Rat brain cortex was collected, and 6 sites from 5'-cap to 3'-end in GAPDH mRNA sequence were measured by SYBR Green I real-time RT-PCR to determine the degradation of GAPDH mRNA in brain cortex. Postmortem intervals were extended to 0, 1, 5, 9, 12 days. RESULTS: All the Ct values of GAPDH mRNA at different sites from the 5'-mRNA cap structure to the 3'-end (GAPDH mRNA 1-GAPDH mRNA 6) in brain correlate with the postmortem periods, while different sites showed different degradation rate. The rates of GAPDH mRNA 4-6 degradation were lower than those of 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. However, the relation between GAPDH mRNA 4-5/GAPDH mRNA 6 ratio and postmortem periods were not observed. CONCLUSION: Different sites of housekeeping gene GAPDH in the same tissue share different degradation rates. The degradation of those sites near to 3'-end of mRNA show more precise to deduce advanced stage PMI.

[The degradation of housekeeping mRNA in dead rats by real-time RT-PCR].

OBJECTIVE: To explore the feasibility of real-time RT-PCR of housekeeping mRNA degradation in dead rats in order to seek new suitable techniques for post-mortem interval (PMI). METHODS: The levels of GAPDH mRNA and beta-actin mRNA in the brain and spleen of the rats were measured at different times after death by the SYBR Green I real-time RT-PCR. The relative mRNA level was indicated with the Ct value, then the relationship between the Ct value and PMI were analyzed and the corresponding regression equation was obtained. RESULTS: The Ct values of GAPDH mRNA and beta-actin mRNA by SYBR Green I real time RT-PCR correlated well with the PMI. CONCLUSION: The SYBR Green I real-time RT-PCR is a reliable technique for studying mRNA degradation. Adoption of the housekeeping genes eliminates systematical errors of other genes which have individual difference. As an objective and dynamic indication, Ct value has a good correlation with PMI, and could be applied for estimating PMI, especially in late period.