| Objective:To investigate the expression of skeletal troponin I(sTnI) mRNA changes induced by contusion in rat skeletal muscle using Real time fluorescence quantitative polymerase chain reaction , aimed to provide some help for forensic estimation of wound age.Methods:A total of 63 Sprague-Dawley male rats, around 10 to 12 weeks old, weighing between 280 and 300 g,were used in this study. All procedures were approved by the Animal Center of Shanxi Medical University. The rats were divided into 3 parts : A total of 63 Sprague-Dawley rats were divided into control group (n=6) and 0.5, 1, 6, 12, 18, 24, 30 and 36 h (n=6) contusion groups, and another 9 rats were divided into 0.5, 1, and 6 h groups which received contusion injury after death.After the rats were anesthetized with ethylether and the right posterior limb was shaved, a depilatory agent (self-made) was applied to remove residual hair. Subsequently, the rats were fixed, and a 250g counterpoise was raised and allowed to fall freely 150cm through a clear Lucite guide tube onto the right posterior limb of rats. The samples were extracted respectively and stored in liquid nitrogen. Total RNA were isolated from skeletal muscle of three groups, and reverse transcription polymerase chain reaction was conducted to synthesize the 1-st strand cDNA. Then standard curves were made for sTnI and house keeping gene (RPL32) with cDNA. With the use of sequence-specific primers, the expression level of sTnI mRNA was studied by SYBR Green I Real Time PCR.Results:(1)The purity,concentration and integrity of Total RNA were evaluated by Agilent 2100 bioanalyzer and 2% formaldehyde-denaturing agarose gel electrophoresis. The test results indicated that the Total RNA suitable for the downstream experiment applications.(2) As shown in the standard curve, the amplification efficiency of the two genes was 103.6% and 100.5%, respectively. The inflection points and baseline of amplification plots were distinct and evenness respectively;The dissociation curves of the two genes showed signal cusps, and the dissociation temperatures were 88°C and 84°C, respectively. The results could be explained by that there were no other PCR productions besides our target genes and there was no primer dimmer.(3)Our results normalized by Ribosomal Protein L32 (RPL32)showed that the expression levels of sTnI mRNA down-regulated 78.17% (P<0.05), 41.58% (P<0.05) and 32.13%(P<0.05) at 0.5, 1 and 6 h after contusion compared to 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. (4) Although no change in the expression levels of sTnI mRNA was observed between the normal skeletal muscle and the control, a significant decrease in the expression levels of sTnI mRNA in the injured skeletal muscle was noted when compared with those in the control and normal skeletal muscle (P < 0.05).(5) 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. Conclusions:(1) A significant decrease in the expression levels of sTnI mRNA in the injured skeletal muscle was noted when compared with those in the control and normal skeletal muscle (P < 0.05),suggesting that the non-injured skeletal muscle from the same contused rat could be used as a control to evaluate the contused muscle.(2) The time-dependent expression of sTnI mRNA after contusion within 36hs was potentially indicative for estimation of early wound age.(3) The merits of the Real time fluorescence quantitative polymerase chain reaction , such as its good accuracy, sensibility and repeatability, enable it as one of the most important tools in the research of forensic medicine.
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