【摘要】 目的 :探讨聚丙烯酰胺凝胶银染技术在半定量RT PCR中的应用。方法 :提取正常组大鼠和链脲佐菌素致糖尿病模型组大鼠心肌组织总RNA ,逆转录得到cDNA第一链 ,以此为模板进行PCR扩增 ,利用聚丙烯酰胺凝胶电泳和DNA银染技术分析不同循环数及模板量与PCR产物量间的关系。结果 :PCR扩增反应在第 2 0～ 2 5个循环 ,起始模板量为 0 8～ 2 4 μg时 ,PCR产物量与起始模板量呈现较好的线性关系。 结论 :通过控制起始模板量 ,减少PCR循环数使目的基因的扩增处于PCR扩增指数期 ,利用聚丙烯酰胺凝胶电泳和DNA银染技术 ,可以通过比较PCR扩增产物量的变化以分析目的基因表达水平的差异更多还原

【Abstract】 AIM:To analyze the application of silver staining of polyacrylamide gels in semi quantitative RT PCR;METHOD:Total RNA was extracted from myocardium tissue of normal group and diabetes mellitus model group induced by streptozotocin,and the gene of glucose transporters 4(Glu T4) and β actin were amplified by RT PCR.The effects of different cycles and different quantities of templates on the quantities of PCR products were evaluated respectively.RESULT:With the methods of silver staining of polyacrylamide gels,PCR products could be detected after 16 cycles in PCR amplification; In cycle 25 th ,when the quantities of template are between 0.8～2.4 μg,the rate of target gene products vs inner check products shows linear relationship with the quantities of template.CONCLUSION:The level of mRNA expression of target gene could be revealed by silver staining of polyacrylamide gels in semi quantitative RT PCR.更多还原