培养乳鼠心肌细胞时钟基因RT-PCR 检测方法的建立

目的 建立检测培养乳鼠心肌细胞中时钟基因的RT-PCR 方法。 方法 根据GeneBank 基因库大鼠时钟基因bmal1、per2 和dbp 全序列设计合成了3 对寡聚核苷酸引物。以离体培养的乳鼠心肌细胞中提取RNA 为模板, 筛选了(PCR) 最佳反应条件, 并进行了特异性检验。 结果 在20 μl 体积中, PCR 最佳系统组成分别为:dbp:Taq 酶、dNTP 和Mg²⁺的用量分别为0.5 U、0.006 μmol 和0.03 μmol;最佳退火温度为58 ℃, 循环30 次;bmal1:Taq 酶、dNTP 和Mg²⁺ 的用量分别为0.5 U、0.006 μmol 和0.035 μmol;最佳退火温度为57 ℃, 循环30 次;per2:Taq 酶、dNTP 和Mg²⁺的用量分别为0.5 U、0.006 μmol 和0.05 μmol;最佳退火温度为58 ℃, 循环32 次。 结论 成功建立RT-PCR 检测大鼠离体培养心肌细胞时钟基因的方法。

Establishment of RT-PCR for detecting clock genes in cultured rattus cardiac myocytes

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.