关键词: 聚合酶链式反应, 时钟基因, 心肌, 大鼠, 细胞培养

Abstract: AIM: To establish a PCR method for investigating the expression of clock genes in cultured rattus cardiac myocytes. METHODS: PCR was carried out using 3 primer pairs based on the published sequences of dbp, bmal1 and per2 genes of rattus.The conditions of PCR were optimized and the specificity of amplication was tested. RESULTS: In a volume of 20 μl, the optimal PCR mixture of bmal1 gene contains 0.5 U Taq polymerase, 0.006 μmol dNTP and 0.035 μmol Mg²⁺;the annealing temperature being 57 ℃;and circle times being 30.In a same volume, the optimal PCR mixture of dbp gene contains 0.5 U Taq polymerase, 0.006 μmol dNTP and 0.03 μmol Mg²⁺;the annealing temperature being 58 ℃;and circle times being 32.The optimal PCR mixture of per2 gene contains 0.5 U Taq polymerase, 0.006 μmol dNTP and 0.05 μmol Mg²⁺;the annealing temperature being 57 ℃;circle times being 30.The specificity of amplication was very high. CONCLUSION: The PCR method can successfully detect mRNA expression of clock genes in cultured rattus cardiac myocytes.

Key words: PCR, clock gene, cardiac myocyte, rattus, cell culture