Application of ITO Method in Determination of Special Half-Sibling Relationship.

OBJECTIVES: To identify whether the relationship between Zhang A, Zhang B, Zhang C and Zhang X is the half-sibling relationship whose mother is sister (hereinafter referred to as the special half-sibling relationship) or the common first cousin relationship and discuss the application of ITO method in discriminating the special kinship. METHODS: DNA was extracted from blood stain of four identified individuals, PowerPlex® 21 System and AGCU 21+1 STR kit were used to detect autosomal STR genetic markers. Investigator® Argus X-12 QS kit was used to detect the X chromosome STR genetic markers, the special half-sibling index (SHSI) and first cousin index (FCI) and their likelihood ratio (LR) were calculated by ITO method. RESULTS: The LR results of SHSI to FCI, which were calculated based on autosomal STR genotyping and the analysis of X-STR genotyping results suggested that the relationship between Zhang A, Zhang B, Zhang C and Zhang X was inclined to be a special half-sibling relationship. CONCLUSIONS: For the identification of special kinship, it is necessary to comprehensively apply various genetic markers according to the case. After the conclusion that shared alleles cannot be excluded from the analysis, ITO method can be further used to establish discriminant assumptions according to the specific case to obtain objective and reliable identification opinions.

[Estimation of early postmortem interval using beta-actin mRNA in rat's brain, heart and kidney].

OBJECTIVE: To explore the relationship between beta-actin mRNA degradation in SD rat's brain, heart and kidney and early postmortem interval (PMI) in order to find new markers for estimating early PMI. METHODS: Rats were sacrificed and kept in the place at a temperature of 20 degrees C. The total RNA were extracted from the brain, heart and kidney at different PMI points. Real time RT-PCR was applied to determine beta-actin mRNA levels in total RNA and the results were given in the form of Ct values. Linear relationships between PMI and Ct values were obtained and the functions of linear regression were established. RESULTS: The great decrease of beta-actin mRNA level were observed in the three organs. The degradation rate was obviously higher in 24 hours after death in the heart and kidney. However, there were no significant changes in the brain. The changes of Ct values and PMI showed a good linear relationship. CONCLUSION: beta-actin mRNA in rat's brain, heart and kidney degrades obviously after death and can be used for estimating early PMI by its degradation rules.

[Effects of triptolide-medicated serum on secretion function of adrenocortical cells isolated from rats].

OBJECTIVE: To study the effects of triptolide-medicated serum on secretory function of adrenocortical cells isolated from rats. METHODS: Thirty SD rats were randomly divided into control group, prednisone group, and low-, medium- and high-dose triptolide groups. Rats were administered with normal saline, prednisone and low-, medium- and high-dose triptolide respectively by gastrogavage to prepare sera containing drugs. Primary adrenocortical cells were isolated from normal male rats and cultured with sera containing drug for 48 hours. Expression of proliferating cell nuclear antigen (PCNA) was observed by immunohistochemical method and number of PCNA-positive cells was counted. Ultrastructure of adrenocortical cells was observed under a transmission electron microscope. Content of corticosterone in supernatant of adrenocortical cell culture was detected by enzyme-linked immunosorbent assay, and real-time fluorescence quantitative polymerase chain reaction (PCR) was employed to investigate the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA. RESULTS: As compared with the control group, content of corticosterone in supernatant of adrenocortical cell culture and expression of 3beta-HSD mRNA were significantly increased in the triptolide-treated groups, and the numbers of PCNA-positive cells were increased in the medium- and high-dose triptolide groups, however, they were decreased in the prednisone group. CONCLUSION: Triptolide-medicated serum can increase the secretion of corticosterone in rat adrenocortical cells in vitro.

[Estimation of postmortem interval using microRNA and 18S rRNA degradation in rat cardiac muscle].

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.