课题背景：本课题为吉林省科学技术发展计划项目（200505216）。agrin蛋白聚糖在多种组织广泛分布，其对轴突生长有重要作用，是神经肌肉接头形成、维持的必要条件。此前agrin mRNA定量检测包括常规聚合酶链反应法和Taqman探针法。常规聚合酶链反应法只能确定同一批上样样本的相对比例，且聚合酶链反应需要逐一调整待测样本量方能克服平台效应并保证聚合酶链反应扩增产物生成量在电泳能够检测的范围内，很难完成。现有的Taqman探针法除成本极高外，上下游引物存在多个连续互补碱基，应用有局限。

术语解析：实时定量聚合酶链反应：在聚合酶链反应体系中加入荧光基团，利用荧光信号积累实时监测整个聚合酶链进程，最后通过标准曲线对未知模板进行定量分析的方法 。

同行评价：实验设计合理、资料完整、论证全面、步步深入的从各方面证明和规范了一套完整的检测方法，对相关研究很有参考价值。

摘要
目的：agrin基因对神经肌肉接头的形成、维持起着决定作用，也对神经元轴突的形成、延长、维持有重要作用，其表达产物在机体内分布广泛。设计绝对定量嵌合荧光法检测大鼠agrin基因mRNA的实时定量聚合酶链反应方法，并检测其特异性、敏感性和可重复性。
方法：实验于2007-04/11在吉林大学基础医学院三综合实验室和吉林大学第一医院传染科实验室完成。以Primer 3软件设计大鼠agrin基因、ACTB基因mRNA嵌合荧光实时定量聚合酶链反应检测引物，经检索Blast验证特异性，并以Primer 5设计构建agrin基因标准品引物。选择清洁级Wistar大鼠交配，获取3日龄乳鼠，取脑，获取mRNA、cDNA，常规反转录-聚合酶链反应获取agrin 554个碱基DNA片断。PCR填加T7启动子后，T7 RNA Polymerase合成agrin基因标准品，消化模板。分光光度计测定其浓度，1∶5倍比稀释3次；经反转录反应合成agrin基因cDNA标准品，测序验证特异性。以ACTB引物反转录cDNA，1∶5倍稀释3次，构建ACTB基因标准品。常规聚合酶链反应确定、优化反应条件。标准曲线确定实时定量聚合酶链反应标准曲线的相关性，融解曲线验证检测方法的特异性，作重复性检测。
结果：①agrin基因有意义链引物序列：GAA GGC CAG GTG CGA ATC A，反义链引物序列：CAC AGG TAG CAG TGG AGC CAA G，内标：ACTB基因有意义链引物序列：GGA GAT TAC TGC CCT GGC TCC TA，反义链引物序列：GAC TCA TCG TAC TCC TGC TTG CTG，优化反应条件：95 ℃ 变性5 s，60 ℃ 退火20 s，72 ℃ 延伸20 s。②agrin基因、ACTB基因标准品所获得的标准曲线确定实时定量聚合酶链反应标准曲线的相关性均 > 0.95。③聚合酶链反应产物电泳、测序、实时定量聚合酶链反应融解曲线证明特异性高、重复性好、灵敏度达到精确定量要求。
结论：绝对定量嵌合荧光法检测大鼠agrin基因mRNA的实时定量聚合酶链反应方法可以作为大鼠agrin基因mRNA检测的标准方法。
关键词：实时定量PCR；agrin基因；ACTB基因；大鼠；组织构建

郭阳，江新梅，郭会敏，刘永茂，王峰，施雨露，冯相伟，朱武飞，邢英琦，赵清石，马学玲，杨蕴天，杨绿. 大鼠agrin基因实时定量聚合酶链反应的检测方法[J].中国组织工程研究与临床康复，2008，12(7):1259-1263
[www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1259(ps).pdf]

Real-Time PCR method to detect rat agrin mRNA

Abstract

AIM：agrin plays a critical role in myoneural junction formation and maintenance, and also regulates neurite formation, elongation, and maintenance. agrin expression products distribute extensively in body. In this study, we designed a perfect Real-Time PCR method to quantitate rat agrin mRNA and test the specificity, sensitivity and repeatability of this method.
METHODS: The experiment was performed at the Third Combined Laboratory of Jilin University and the Laboratory of Department of Infectious Disease of Jilin University from April to November 2007. The perfect Real-Time PCR primers to detect rat agrin mRNA and rat ACTB mRNA were designed by Primer3 software. After the specificity of these primers was proved in Blast, the primer to construct standard preparation of rat agrin mRNA was designed by Primer5 software. Wistar rats (clean grade) were selected and mated to get neonate rat. After operation, brain tissue of 3-day neonate rat was used for mRNA harvest. The reverse transcription-polymerase chain reaction was performed to obtain 554 bp DNA fragment of rat agrin. After T7 promotor was connected to rat agrin DNA by PCR, standard preparation of rat agrin mRNA was constructed by T7 RNA Polymerase. DNA was digested and the concentration of standard preparation of rat agrin mRNA was detected by spectrophotometer. Rat agrin mRNA was diluted by multiproportion of 1∶5 for 3 times, and standard preparation of rat agrin cDNA was obtained by reverse transcription reaction. The standard preparation of rat ACTB mRNA was constructed by reverse transcription reaction while the perfect Real-Time PCR primer was used. mRNA was diluted by multiproportion of 1: 5 for 3 times, and standard preparation of rat ACTB cDNA was obtained. Reaction conditions were confirmed and optimized using PCR proceeding. The standard preparations were sequenced to test theirs specificity. Dependablity of standard curve, specificity of dilapsus curve and repeatability of the method were detected.
RESULTS: ①The primer of Real-Time PCR was set: Rat agrin mRNA sense GAA GGC CAG GTG CGA ATC A, antisense CAC AGG TAG CAG TGG AGC CAA G; internal standard: Rat ACTB mRNA sense GGA GAT TAC TGC CCT GGC TCC TA, antisense GAC TCA TCG TAC TCC TGC TTG CTG. Optimized PCR proceed was an initial denaturation cycle of 5 seconds at 95 ℃, followed by 40 cycles (5 seconds at 95 ℃, 20 seconds at 60 ℃ and 20 seconds at 72 ℃). ②The rate of dependablity about the standard curves of rat agrin mRNA and rat ACTB mRNA >0.95. ③Electrophoresis, sequencing result of PCR products and dilapsus curve of Real-Time PC verified the specificity of the method. The repeatability of the method was good, and the sensitivity met the requirements.
CONCLUSION: The perfect Real-Time PCR method to quantitate rat agrin mRNA is tested and can be used as a standard method.

Guo Y, Jiang XM, Guo HM, Liu YM, Wang F, Shi YL, Feng XW, Zhu WF, Xing YQ, Zhao QS, Ma XL, Yang YT, Yang L. Real-Time PCR method to detect rat agrin mRNA.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008; 12(7):1259-1263(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1259(ps).pdf]