权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of easy and quantitative reverse transcription-polymerase chain reaction method (MRT-PCR) for minute clinical samples and its clinical applications

针对微小临床样本的简易定量逆转录聚合酶链式反应方法（MRT-PCR）的开发及其临床应用

基本信息

批准号：
06557036
负责人：
WATANABE Tsuyoshi
金额：
$ 7.68万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

We developed a new reverse trascription-polymerase chain reaction method (MRT-PCR) for acurate quantitation of mRNA expression of specific gene in minute samples such as biopsy specimens and micro-dissection samples from kidneys of experimental model animals ; A point mutation which newly create or lose a restriction enzyme cleavage site in a RT primer, so that products in PCR reaction in which both genomic DNA and cDNA are equally amplified using the same primers. The PCR product from genomic DNA and that from cDNA can be differenciated by a specific restriction enzyme treatment. Advantages of this method are as follows ; this method has less chance of error based on different sensitivity of Taq polymerase reaction for primers compared to so-called competitive RT-PCR method in which a mutate d competiter primer and original primers are simultaneously used in PCR reaction, and this provides the mRNA quanyity per a single cell comparing content of PCR product derived from genomic DNA and that from mRNA without measuring protein or nucleic acid contents in minute samples. We have applied this method for detection of quantitative distribution of EP3 subtype of prostaglandin E receptor, platelet-activating factor receptor, clusterin and thromboxane A_2 receptor in micro-dissected nephron of normal and disease model animals. Moreover, we recently developed another new MRT-PCR method for human vitamin D receptor in which restriction fragment length polymorphism (FRLP) can be analyzed simultaneously as mRNA content relative to genomic DNA.This methods are being applied for trials for early detection of high risk grop for bone diseases in hemodialysis patients. Our new MRT-PCR method is believed to be applied for wide ranges of clinical and basic medical fields

我们建立了一种新的逆转录-聚合酶链反应（RT-PCR）方法，用于精确定量检测实验模型动物肾脏活检标本和显微解剖标本中特定基因的mRNA表达;在RT引物中新产生或失去限制酶切割位点的点突变，从而在PCR反应中使用相同的引物同等地扩增基因组DNA和cDNA的产物。基因组DNA的PCR产物和cDNA的PCR产物可通过特异性限制性内切酶处理区分。这种方法的优点如下：与在PCR反应中同时使用突变的竞争引物和原始引物的所谓竞争性RT-PCR方法相比，该方法具有较少的基于Taq聚合酶反应对引物的不同灵敏度的错误机会，并且这提供了每单个细胞的mRNA数量，比较了来自基因组DNA的PCR产物的含量和来自mRNA的含量，而没有测量微量样品中的蛋白质或核酸含量。我们应用此方法对正常和疾病模型动物肾单位中前列腺素E受体、血小板活化因子受体、聚集素和血栓素A_2受体的EP_3亚型进行了定量检测。此外，我们最近开发了另一种新的人维生素D受体的MRT-PCR方法，其中限制性片段长度多态性（FRLP）可以同时分析mRNA相对于基因组DNA的含量，这种方法正在应用于血液透析患者中骨疾病高危人群的早期检测试验。本研究建立的新方法可广泛应用于临床和基础医学领域