单细胞逆转录聚合酶链反应与膜片钳同步技术

Single cell reverse transcriptase polymerase chain reaction methods combining with patch clamp techniques

目的 建立与膜片钳技术相结合的单细胞逆转录聚合酶链反应(reversetranscriptasepolymerasechainreaction,RT -PCR)技术。方法 采用传统酶解法分离出SD大鼠单个心室肌细胞,应用全细胞方式膜片钳技术记录通道电流后对同一细胞吸取内容物,采用RT-PCR技术,分别对单细胞内GAPDH及L型钙通道的mRNA表达进行逆转录聚合酶链反应。结果 电泳结果显示GAPDH及L型钙通道条带,对后者的测序分析结果与cDNA基因文库符合率为 100%。结论 此实验方法可以用于同时进行膜片钳操作及单细胞RT-PCR检测。

Objective The aim of this study is to establish the method of single cell reverse transcriptase polymerase chain reaction methods(RT-PCR)combining with patch clamp techniques Methods Single ventricular myocytes were isolated enzymatically using Langendorff-perfusion apparatus.After recording the current,plasm was inhaled into a pipette which contain DEPC(Diethypyocarbonate)water.The mRNA level of GAPDH and L-type calcium channels were measured using RT-PCR in single cell.Results The bands were showed by electrophoresis.Sequencing results were same as the cDNA library.Conclusion It is feasiblely to bind single cell RT-PCR methods with patch clamp techniques.