人类SUZ12基因实时荧光定量PCR检测方法的建立
SUZ12;SYBRGreenI;实时荧光定量PCR,陈凤花,胡丽华,王琳,李一荣,周志明,陈凤花,通讯作者:,QuantificationofhumanSUZ12withSYBRGreenIreal-timefluorescentpoly
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陈凤花, 胡丽华, 王琳, 李一荣, 周志明, 华中科技大学附属协和医院检验科 湖北省武汉市 430022
陈凤花, 2006年华中科技大学同济医学院博士毕业, 主治医师, 主要从事肿瘤的实验诊断研究.
湖北省科技攻关项目, No. 2005AA304B08
通讯作者: 陈凤花, 430022, 湖北省武汉市, 武汉协和医院检验科. chfh100@126.com
电话: 027-85726367
收稿日期: 2006-11-01 接受日期: 2007-01-01
Quantification of human SUZ12 with SYBR Green I real-time fluorescent polymerase chain reaction
Feng-Hua Chen, Li-Hua Hu, Lin Wang, Yi-Rong Li, Zhi-Ming Zhou
Feng-Hua Chen, Li-Hua Hu, Lin Wang, Yi-Rong Li, Zhi-Ming Zhou,Laboratory Department and Institute of Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China
Supported by:The Key Program of Science and Technology Foundation of Hubei Province, No. 2005AA304B08
Correspondence to:Feng-Hua Chen, Department &Institute of Immunology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China. chfh100@126.com
Received:2006-11-01 Accepted:2007-01-01
Abstract
AIM: To establish a real-time fluorescent polymerase chain reaction (PCR) for quantifying human SUZ12.
METHODS: The SUZ12 fragment in pure form from classical RT-PCR was cloned to pGEM-T vector, and recombinant plasmid pGEM-SUZ12 was purified and quantified by spectrophotometry. A standard curve was established using a serial dilution of quantified plasmids to measure SUZ12, using SYBR Green I real-time fluorescent PCR, and the characteristics of the specific SUZ12 amplicon were analyzed by the melting curve.
RESULTS: The method could detect as few as 14.2 copies. Good linearity was found from 14.2 to 1.42 × 108 copies/reaction, and the correlation coeffecient was -1.00. The intra- and interassay variation of 1.42 × 104 copies/reaction was 1.8% and 2.8%, respectively. Melting curve analysis showed a single peak, and melting temperature (Tm) was (81 ......
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