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

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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