实时定量RT-PCR方法检测豚鼠TSHR免疫BALB/c小鼠甲状腺TSHR的表达  

Real-time RT-PCR for detection of expression of TSHR mRNA in thyroid of BALB/c mice immunized by guinea pig TSHR

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,天津300070 [2]天津医科大学病理生理教研室

出　　处：《天津医科大学学报》2005年第4期509-511,514,共4页Journal of Tianjin Medical University

基　　金：国家自然科学基金资助(编号:30370682);天津市科技发展计划项目(编号:05YFGDSF02700)

摘　　要：目的:建立外标准法SYBRGREENI实时定量(real-time)RT-PCR方法,以检测豚鼠TSHR分子免疫对BALB/c小鼠甲状腺TSHRmRNA的表达的影响。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,得到333bp扩增产物。经回收纯化后作为real-timePCR的标准品,稀释为106、105、104、103、102、101拷贝,制作标准曲线;并同时进行普通PCR,比较其灵敏度。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方法可以灵敏的检测到10copy的核酸,灵敏度为普通PCR的104倍;溶解曲线只有特异峰,溶解温度为82.9℃。没有明显的引物二聚体和非特异峰出现。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SYBRGREENI实时定量RT-PCR具有灵敏度高,特异性强,重复性好的特点,比普通PCR更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 to detect the expression of TSHR mRNA in thyroid in 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 is 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 is performed at the same time to compare the sensitivity of the technique. The in- ter-coefficient variations of different standard points are 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 control （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：外标准法实时定量RT—PCR TSHR MRNA SYBR GREEN I 

分 类 号：Q451[生物学—生理学] Q786