| Objective: Differential display was used to isolate early time-related genes from the contused skeletal muscle of rats in order to find one or several sensitive gene, and the expression levels of the differential display genes mRNA in skeletal muscle were examined by real-time PCR to determine whether it can be used as a marker for wound age estimation.Methods: 1. A total of 12 Sprague-Dawley male rats, around 10 to 12 weeks old, weighing between 250 and 300 g, were used in this study. The rats were divided into 2 parts: control group (n=6) and contusion group (n=6). Animals were kept under a 12 h light-dark cycle with free access to food and water. After the rats were anesthetized with ethylether and the right posterior limb was shaved, a depilatory agent was applied to remove residual hair. Subsequently, the rats were placed on a foam bed, and a 250 g counterpoise was raised and allowed to fall freely 150 cm through a clear Lucite guide tube onto the right posterior limb of rats. After injury was made, the rats were reared in a cage and fed commercial rat food and tap water ad libitum. At 4 h after contusion, the rats were sacrificed with a lethal dose of pentobarbital (350 mg/kg of body weight intraperitoneal injection). Approximate 100 mg muscle sample was dissected from the right posterior limb, cut into two parts and immediately frozen in liquid nitrogen. Total RNA was isolated from muscle specimens (weighing approximately 50 mg) using the SV Total RNA Isolation System following the instructions provided with the kit. The concentration (ng/μL) and total RNA integrity was assessed using an Agilent 2100 Bioanalyser by loading samples onto the Eukaryote total RNA nano-chip. And only those RNA with a RNA Integrity Number (RIN) above 8.0 and with clearly visible 28/18s peaks was used for the following experiment.2. A total of 12 combinations of anchor primers and random primers were used to amplify the cDNA from the control group and contused group skeletal muscle. The PCR products were analyzed by the PAGE with silver staining. The differential display bands were purified with the vector, cloned, sequence analyzed and homologated with GenBank.3. The specific primers to the differential dispaly fragments were used to amplify the specific PCR products from the total RNA by the Real-time PCR, in order to indentify the authenticity of differential display fragments.4. In order to identify the endogenous reference genes for normalization of the gene expression in the contused skeletal muscle, a panel of nine candidate reference genes, such asβ-Actin and GAPDH, were tested in the control group, 6 h and 12 h group after contusion by the Real-time PCR. Then the potential endogenous reference genes were tested by three different software packages, Normfinder, geNorm and BestKeeper.5. A total of 54 Sprague-Dawley male rats, which were divided into 2 parts : control group (n=6) and 0.5, 1, 6, 12, 18, 24, 30 and 36 h (n=6) contusion group, were used to examine the expression levels of the differential display genes mRNA in skeletal muscle by real-time PCR.Results: 1.All samples were performed on Agilent 2100 Bioanalyzer using the RNA 6000 LabChips Kit. Approximate 0.1-0.4μg of total RNA was extracted from each milligram wet muscle using the SV Total RNA Isolation System. And only those RNA with a RNA Integrity Number (RIN) above 8.0 and with clearly visible 28/18s peaks was used for the following experiment.2. A total of 21 differential display bands were cut from the PAGE, and 12 of them were successfully amplified. Then the DNA was recombined to the vector, and cloned in the E.coli. The sequence was analyzed by the Takara. As a result, 5 fragments were long enough to be homologated with GenBank.3. Each of 5 fragments was successfully amplified from the total RNA using the special primers. And the dissociation curve of them all showed singal cusps, which suggested that there were no other PCR productions besides our target gene, and there was no primer dimmer.4. The results, which were analyzed by the Normfinder, geNorm and BestKeeper, showed that the RPL13 and RPL32 were more stable than other endogenous reference genes in normalizing the genes expression of skeletal muscle.5. The expression level of sTnI mRNA decreased gradually after contusion, at 0.5 h , 1 h and 6 h after contusion, the expression level decreased to 78.17% (P<0.05), 41.58% (P<0.05) and 32.13% of that in the control group. The expression level of Mx2 mRNA upgraded to 84.3 times (P<0.05) of that in the control group at 0.5 h after contusion, then it decreased gradually, at 18 h the expression level was 0.65 times of that in the control group. At 24 h after injury there was another peak of expression which reached 37.1 times of that in the normal control group. The expression level of Frag-3 mRNA upgraded to 243.5 times of that in the normal group at 6 h after contusion, then it decreasd to normal expression level. At 18 h after injury, there was another peak of expression which was 6.3 times of that in the normal group, and then it again decreased to normal expression level. The expression level of Cox6c mRNA had a lesser change. It varied from 0.24 to 1.57 times of that in the normal group. The highest expression level was 1.57 times of that in the control group at 0.5 h after contusion, and the minimum expression level was 0.24 times of that in the control group at 30 h after contusion. The expression level of Frag-5 mRNA was high during 36 h after contusion. At 0.5 h , 1 h and 6 h after contusion, the expression level upgraded to 5.7, 6.1 and 4.27 times of that in the control group . The highest expression level was 8.6 times to that in the control group at 18 h after contusion. At 24 h, 30 h, and 36 h after injury, the expression level decreased to 2.9, 2.6 and 1.8 times of that in the control group.Conclusion: Five fragments were found by DDRT-PCR. Three of them had a high homology with the gene sequence of sTnI, Mx2 and Cox6c, and the other two fragments may be new genes. sTnI mRNA and Cox6c mRNA changed regularly along with the injury time, and could be considered as new markers of wound time estimation. |
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