MRNA Differential Display Technology In Skin Contusion Application

Objective: To use the silver staining technology showing the differences in rat's skin injury-related genes, aimed at finding one or more of the sensitive and injury-related genes, to infer the time in practice of forensic pathology to provide scientific, effective reference.Methods: Experiment was divided into the normal group(n=6)and experimental group(n=6).Experimental group:intraperitoneal injection of pentobarbital sodium anesthetized rats, conventional surgical sterilization, with 250 g in the hammer 150 cm high degree of free fall, resulting in the quadriceps right hind legs skin contusion.after 4 h contusion,rats were killed from neck, skin tissue was extracted;Control group:the rats were killed from neck, skin from the corresponding tissue. Total RNA was extracted from the control group and the experimental group. after the quality evaluation, the polymerase chain reaction was done. Four sets of anchor primers and three sets of arbitrary primers, total twelve primers combinations were used for DD-RTPCR amplification. after 1.5% agarose gel electrophoresis and 8% polyacrylamide gel electrophoresis(400 V, 3 h), the differences of the products were screened. Polyacrylamide gel was on a fix, washing, dyeing, color, termination, and rinse. The different bands were cut with sterile blades and reamplified. the amplified products by purification were connected to pMD18-T Vector and transfered into JM109 competent cells,then trained in the SOC medium and LB agar plate medium (Amp+). After being cloned, all DNA fragments were sequenced, the sequencing results were compared with GenBank database for homology analysis. Results: (1)Total RNA meet the quality requirements and the A260/A280 value was up to 1.913.(2)There were different bands between experimental group and control group after 1.5% agarose gel electrophoresis.(3)There were different bands between experimental group and control group after 8% polyacrylamide gel electrophoresis. There were total 22 different bands between 150-600bp. (4)The reamplified products were connected to pMD18-T Vector and transfered into JM109 competent cells,then trained in the SOC medium and LB agar plate medium (Amp+) that appeared blue and white colonies colony.(5)Five different bands were found: troponin,the DNA-binding protein,cytochrome c oxidase,Rattus norvegicus myxovirus (influenza virus) resistance 2,Mouse DNA sequence from clone RP23-403G13 . Conclusions:A variety of gene expression participate in the process of skin injury. Troponin and cytochrome c oxidase mRNA expression increased after inury, But it was unclear that the injury is a protective effect or damage, whether there is a certain amount of time relations have yet to be studied further.