RESUMO
OBJECTIVE@#To explore the stability of internal controls in human cardiac muscle by real-time RT-PCR during early postmortem interval (PMI) in order to find the most stable marker.@*METHODS@#Ten individuals with similar environmental conditions (the average store temperature: 25 degrees C) and different PMI ranging from 4.3 to 22.3 h were selected. Total RNA was extracted from each sample and six commonly internal controls were used including beta-actin, GAPDH, B2M, U6, 18S rRNA and HSA-miR-1, and the expression was detected in cardiac muscle by real-time RT-PCR. The expression stability of internal controls was evaluated using genormPLUS software during early PMI. The internal control with the most stability was selected. The relationship between the most stable marker and its expression level affected by some other parameters such as age, gender and cause of death was also analyzed.@*RESULTS@#The U6 showed the most stable expression during early PMI in cardiac muscle, and its expression level was not affected by those parameters including age, gender and cause of death (P > 0.05).@*CONCLUSION@#U6 may be a valuable internal control for the study of relationship between PMI determination and degradation of nucleic acid in human cardiac muscle by real-time RT-PCR.
Assuntos
Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Actinas/metabolismo , Causas de Morte , Estudos de Avaliação como Assunto , Patologia Legal/métodos , MicroRNAs/metabolismo , Miocárdio/metabolismo , Mudanças Depois da Morte , RNA/metabolismo , Estabilidade de RNA , RNA Ribossômico 18S/metabolismo , RNA Nuclear Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fatores de TempoRESUMO
OBJECTIVE@#To investigate the degradation changes of beta-actin mRNA and 18S rRNA in different time points and temperature after death, and to explore the relationship between the changes and postmortem interval (PMI) in the brain of mice.@*METHODS@#Twenty-four health adult C57BL/6 mice were randomly divided into two groups (12 each group). They were sacrificed by cervical dislocation and placed in chamber with two different temperature (4 degrees C and 37 degrees C, humidity was 80%). The mice brains were sampled at 6 different time points(immediately, 0.5h, 2h, 6h, 24h, 48h), and total brain RNA were extracted. Ct value of each sample was obtained using RT-PCR and real-time PCR technology, and beta-actin mRNA and 18S rRNA content ratio was calculated. The correlation between the content ratio and PMI was expressed using statistical regression analysis.@*RESULTS@#At 37 degrees C, RNA degradation rate was faster than 4 degrees C, which showed that there was correlation between temperature and RNA degradation. Comparing with the stability of beta-actin mRNA, 18S rRNA was more stable.@*CONCLUSION@#The study on degradation of beta-actin mRNA and 18S rRNA in mice brain using real time PCR technology could provide a new theoretical basis for estimation of PMI and would be supplementary to the traditional methods.
Assuntos
Animais , Feminino , Masculino , Camundongos , Actinas/metabolismo , Encéfalo/metabolismo , Medicina Legal/métodos , Camundongos Endogâmicos C57BL , Mudanças Depois da Morte , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise de Regressão , Temperatura , Fatores de TempoRESUMO
OBJECTIVE@#To explore the relationship between the time-dependent level changes of microRNA and 18S rRNA and the different postmortem interval (PMI) in rat cardiac muscle.@*METHODS@#SD rats were sacrificed by cervical dislocation and placed at ambient temperature 25 degrees C with a humidity of 50%. Total RNA was extracted from the rat cardiac muscle at different time points after death. The levels of miR-1-2 and 18S rRNA were examined using real-time PCR in rat cardiac muscle. The results were expressed by cycle threshold (Ct) value to explore relationship between PMI and Ct value, and the regression functions were established to estimate PMI.@*RESULTS@#The miR-1-2 level in rat myocardial tissue showed no significant changes within 120 h after death, and then began to decline. The 18S rRNA level increased gradually within 96 h after death, and then declined slowly. The nonlinear relationships were established between Ct value (18S rRNA), deltaCt value (difference between 18S rRNA and miR-1-2) and PMI. The R2 of conics fitting were 0.9487 and 0.8072, respectively.@*CONCLUSION@#Ct value of 18S rRNA and deltaCt value present a good correlation with PMI, and can be markers for estimating early PMI.
Assuntos
Animais , Masculino , Ratos , Patologia Legal , MicroRNAs/metabolismo , Miocárdio/patologia , Mudanças Depois da Morte , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fatores de TempoRESUMO
OBJECTIVE@#To investigate the expression of 18S rRNA and beta-actin mRNA in bloodstain between 8 and 15 days after death and extrapolate the time of bloodstain formation.@*METHODS@#RNA in dried bloodstain at different times was extracted, then quantified for 18S rRNA and beta-actin mRNA by real-time RT-PCR. The bloodstain formation time was deduced based on the changes of the ratio of 18S rRNA to beta-actin mRNA at different time points.@*RESULTS@#The ratio of 18S rRNA to beta-actin mRNA increased gradually with time, indicating that rRNA and mRNA degraded in different rate with time. CONCLUSION; The ratio of 18S rRNA to beta-actin mRNA could be used for estimating the time of bloodstain formation in some period.