外标准法实时定量反转录-聚合酶链反应方法的建立  

Development of real-time RT-PCR with a external standard

作　　者：朱云娟[1] 黄丽娟[2] 陈宁[2] 沈炳玲[2] 陆凤先[2] 

机构地区：[1]天津医科大学寄生虫学教研室,300070 [2]天津医科大学病理生理教研室,300070

出　　处：《中国药物与临床》2006年第1期18-20,共3页Chinese Remedies & Clinics

基　　金：国家自然科学基金资助项目(30370682);天津市科技发展计划基金资助项目(05YFGDSF02700);天津医科大学科学基金资助项目(2005KY13)

摘　　要：目的为了检测BALB/C小鼠甲状腺促甲状腺激素受体(TSHR)表达,建立外标准法实时定量反转录-聚合酶链反应(RT-PCR)方法。方法取正常BALB/C小鼠甲状腺提取甲状腺总RNA,逆转录后进行PCR,PCR产物经回收纯化后作为real-timePCR的标准品,制作标准曲线,优化反应条件使溶解曲线有特异扩增。检测豚鼠TSHR分子免疫的BALB/C小鼠甲状腺TSHRmRNA的表达。结果建立的外标准法SYBRGREENⅠ实时定量RT-PCR方法可以灵敏检测到10copies的核酸,灵敏度为普通PCR的104倍;溶解曲线只有特异峰;不同标准点批间变异系数范围为5.8%￣14.3%(n=6)。与对照组比较,TSHR大分子免疫的BALB/C小鼠甲状腺组织TSHRmRNA水平显著升高(P<0.05)。结论外标准法实时定量RT-PCR具有灵敏度高、特异性强、重复性好的特点,可精确地检测出小鼠甲状腺组织TSHRmRNA的水平。Objective To establish SYBR GREEN I real-time RT PCR method with the external standard in order to detect the expression of TSHR mRNA in thyroid of the mice immunized with guinea pig TSHR. Methods Total RNA prepared from normal BALB/C mice thyroid was reverse transcribed and amplified by PCR. PCR product is a 333 bp DNA fragment. It was purified as the external standard for real-time RT-PCR. Standard curve is formed when standard DNA is diluted at 10^6,10^5,10^4,10^3,10^2,10^1 copies. Ordinary PCR was performed at the same time to compare the sensitivity of the technique. The inter-coefficient variations of different standard points were used to test the reproducibility of the method. The expression levels of TSHR mRNA of thyroid tissue in BALB/C mice injected with guinea pig TSHR molecule are tested with the method. Results It was shown that less than 10 copies TSHR gene could be sensitively detected by this method, which was 10^4 times more sensitive than ordinary PCR. Only a specific peak was shown on the melting curve diagram and the melting temperature was 82.9 ℃. Neither primer dimmer nor non-specific peak was shown on the curve. The inter-coefficient variations of different standard points range from 5.8% to 14.3%（n=6）.The expression levels of TSHR mRNA of thyroid tissue in BALB/C mice injected with guinea pig TSHR molecule were significantly higher than the control group （P〈0.05）. Conclusion It is suggested that real-time RT-PCR with the external standard is a sensitive, specific and reproductive method and it is more accurate than ordinary PCR for determination of TSHR mRNA in thyroid tissue.

关 键 词：逆转录聚合酶链反应 受体 促甲状腺激素 小鼠.近交BALB/C 

分 类 号：R446.9[医药卫生—诊断学]