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

我们建立了一种新的反向转录-聚合酶链式反应(MRT-PCR)方法，用于精确定量实验模型动物肾脏活检标本和显微解剖标本等微小样本中特定基因的mRNA表达：在RT引物中新创建或丢失限制性内切酶切割位点的点突变，从而使在PCR反应中基因组DNA和cDNA的产物使用相同的引物被同等地扩增。通过特定的限制性内切酶处理，可将基因组DNA的扩增产物与来自cDNA的扩增产物区分开来。这种方法的优点是：由于Taq聚合酶反应对引物的敏感性不同，这种方法与竞争RT-PCR方法相比，由于Taq聚合酶反应的不同敏感性而产生错误的机会较少。在竞争RT-PCR方法中，突变的竞争引物和原始引物同时用于PCR反应，从而提供了单个细胞的mRNA质量，可以比较来自基因组DNA的PCR产物的含量，而不需要测量微量样品中的蛋白质或核酸含量。我们应用该方法检测了正常和疾病模型动物肾脏EP3亚型前列腺素E受体、血小板活化因子受体、聚集素和血栓素A_2受体的定量分布。此外，我们最近还建立了另一种新的人类维生素D受体的MRT-PCR方法，该方法可以同时分析限制性片段长度多态性(FRLP)作为相对于基因组DNA的mRNA含量。该方法正在用于血液透析患者骨病高危人群的早期检测。我们的新的MRT-PCR方法被认为可以应用于广泛的临床和基础医学领域

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