Establishment of RT-PCR for detecting clock genes in cultured rattus cardiac myocytes / 中国临床药理学与治疗学

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 ?l, the optimal PCR mixture of bmal1 gene contains 0.5 U Taq polymerase, 0.006 ?mol dNTP and 0.035 ?mol Mg 2+; 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 ?mol dNTP and 0.03 ?mol Mg 2+; 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 ?mol dNTP and 0.05 ?mol Mg 2+; 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.