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|本期目录/Table of Contents|

采用荧光定量RT-PCR法检测超极化激活环核苷酸门控阳离子通道(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2009年第4期
页码:
457-461
栏目:
基础研究
出版日期:
2009-06-25

文章信息/Info

Title:
Qualification of hyperpolarization-activated cyclic nucleotide-gated cation channel mRNA using the fluorogenic quantitative RT-PCR method
作者:
黄维清1; 孔庆暖1; 张亚青2 ; 张七一3
青岛市市立医院:1.病理科，3.心内科，山东 青岛 266071； 2.第四军医大学西京医院病理科，陕西 西安 710032
Author(s):
HUANG Wei-qing1;  KONG Qing-nuan1;  ZHANG Ya-qing2;  ZHANG Qi-yi3
1.Department of Pathology, 3.Department of Cardiology, Qingdao Municipal Hospital, Qingdao 266071, Shandong, China; 2.Department of Pathology, Xijing Hospital, Fourth Military Medical University, Xian 710032, Shaanxi, China
关键词:
聚合酶链反应; 荧光定量; HCN基因; 心脏
Keywords:
polymerase chain reaction;  fluorogenic quantitative;  hyperpolarization-activated cyclic nucleotide-gated cation channel gene;  heart
分类号:
R361.1
DOI:
-
文献标识码:
A
摘要:
目的 建立用SYBR Green荧光染料检测超极化激活环核苷酸门控阳离子通道（HCN4）的实时定量RT-PCR方法。方法 提取窦房结细胞的总RNA，反转录成cDNA后，应用RT-PCR法检测HCN4的表达，通过琼脂糖凝胶回收PCR产物并构建重组质粒，用梯度稀释的质粒模板建立荧光定量RT-PCR法检测HCN4的标准曲线，并检测心脏组织中HCN4的表达水平。结果 成功构建了质粒重组子，并建立用SYBR Green荧光染料在RNA水平检测HCN4通道的标准曲线，相关系数为0.997重复6次实验组内和组间变异系数均<1.0％。心脏组织中窦房结HCN4表达量最高，心室表达量最低。结论 基于SYBR Green荧光染料建立的定量RT-PCR体系敏感性高，线性范围广，重复性好，可快速准确地检测HCN4的表达。
Abstract:
AIM: To establish a fluorogenic quantitative RT-PCR (RTqPCR) method with SYBR Green dye for detecting the expression of hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN4) mRNA. METHODS: Total RNA was extracted from the cardiac sinoatrial node tissue. After reverse transcription, the HCN4 gene was detected using RT-PCR method. PCR products were then gel-purified and subcloned into the PGEM-T Easy vector to construct the recombinant plasmid. The standard curve of the HCN4 gene was established by RTqPCR and used to detect the expression of HCN4 gene in different heart tissues. RESULTS: We successfully constructed the recombinant plasmid and established the standard curve to detect the expression of the HCN4 gene. There was good statistical linear relationship with the regression value 0.997. In each experiment, the intra- and inter-assay coefficients of variation were <1.0%. HCN4 expression was found at the highest level in sinoatrial node and the lowest level in ventricle. CONCLUSION: Real-time RT-PCR based on SYBRGreen dye has been developed with high specificity and sensitivity, which can be well repeated. This method is rapid and can be widely applied for HCN4 gene detection.

参考文献/References

［1］Accili EA, Proenza C, Baruscotti M, et al. From funny current to HCN channels: 20 years of excitation［J］ . News Physiol Sci, 2002, 17:32-37.

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［3］ Chen J, Piper DR, Sanguinetti MC. Voltage sensing and activation gating of HCN pacemaker channels［J］. Trends Cardiovasc Med, 2002, 12(1):42-45.

［4］ Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart［J］. Proc Natl Acad Sci USA, 2003, 100(25):15235-15240.

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［7］ Zagotta WN, Olivier NB, Black KD, et al. Structural basis for modulation and agonist specificity of HCN pacemaker channels［J ］. Nature, 2003, 425(6954):200-205.

［8］ Tollon C, Bedut S, Villeneuve N, et al. Use-dependent inhibition of hHCN4 by ivabradine and relationship with reduction in pacemaker activity［J］. Br J Pharmacol, 2007, 150(1):37-46.

［9］ Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channel dysfunction in a patient with sinus node disease［J］. J Clin Invest, 2003, 111(10):1537-1545.

［10］Fernández-Velasco M, Goren N, Goern N, et al. Regional distribution of hyperpolarization-activated current (If) and hyperpolarization-activated cyclic nucleotide-gated channel mRNA expression in ventricular cells from control and hypertrophied rat hearts［J］. J Physiol, 2003, 553(Pt2):395-405

备注/Memo

备注/Memo:
收稿日期：2008-6-26.基金项目：青岛市科技发展计划立项项目资助(05-2-NS-43) 通讯作者：张七一，主任医师，主要从事心血管疾病研究Email:zqy@public.qd.sd.cn 作者简介：黄维清，副主任医师，硕士Email:huangweiqing_40@163.com
更新日期/Last Update: 2009-06-15