人心房肌KChlP2和SK2基因表达检测及方法研究

The detection of the KChIP2 and SK2 gene expression in human atrial myocardium and method research.

目的检测人心房肌KChIP2和SK2基因表达,并构建相应质粒标准品,为进一步研究基因功能奠定基础。方法提取人心房肌组织总RNA,采用RT-PCR方法获取KChIP2、SK2、GAPDH、β-actin目的片段,与pMD-18Tvector连接成重组质粒,转化到大肠杆菌DH5α中,通过PCR和测序鉴定。提取含目的基因的质粒作为标准品。采用Taqman探针法与SYBR Green I法分别检测人心房肌SK2与KChIP2基因表达和构建标准曲线。结果人心房肌存在KChIP2和SK2基因表达,质粒标准品经过PCR鉴定并测序,与Pubmed数据库完全一致,标准曲线线性关系好,相关系数R2>0.99。结论成功检测人心房肌KChIP2和SK2基因表达,构建的质粒标准品能用于后续实验。

Objective To detect the Expression of KChIP2 and SK2 Gene in Human Atrial Myocardium and construct the plasmid standard for further research on their gene functions.Methods The target fragments of the KChIP2,SK2,GAPDH,β-actin gene were constructed with the total RNA isolated from human atrial myocardium and then was linked with pMD 18-T vector to construct recombined plasmids,which were transformated to Escherichia coli DH5α.Their specificity was tested by direct PCR and sequencing method.The isolated plasmids including the KChIP2,SK2,GAPDH,β-actin gene were used as standards for getting standard curves and detecting the SK2 and KChIP2 gene expression with SYBR Green I and Taqman probe respectively.Results There was KChIP2 and SK2 gene expression in human atrial myocardium.The sequences of the plasmids including the target genes were consistent with the Pubmed database completely.The standard curve has good linear correlation with R2〉0.99.Conclusion We detected the KChIP2 and SK2 gene expression in human atrial myocardium and constructed the recombined plasmid standards successfully,and the plasmid standards can be used for further research.