|Budget Amount *help
¥11,800,000 (Direct Cost : ¥11,800,000)
Fiscal Year 1997 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1996 : ¥4,500,000 (Direct Cost : ¥4,500,000)
Fiscal Year 1995 : ¥4,900,000 (Direct Cost : ¥4,900,000)
The aim of this resaerch project is to develop a technique for in situ detection of mRNA with high specificity and sensitivity, using the amplification of mRNA by the polymerase chain reaction (PCR) method. In the first step of this study, we were able to establish an optimal condition for in situ reverse transcription. Then we further tried to utilize the cRNA,produced by the in situ reverse transcription on tissue sections of the rat brain, as templates for PCR amplification. For this purpose, it is first required to determine the optimal reaction condition of PCR which allows us to amplify cRNAs with the highest efficacy. Namely, 1)the determination of the optimal in situ condition for PCR,2)the examination of efective biotinylation, either by the introduction of biotin into polymerase chain reaction or by the use of biotin-labeled primers, 3)the determination of optimal condition for the visualization of labelled PCR products, and 4)quantitative morphometric analysis using a computor-assisted image analysisincluding the development of a computor-software. Concerning the determination for 1)we were able to obtain generally satisfactory condition. However, the determined condition differed from the known condition for in vitro PCR,and we have been trying to find out the reason for the discrepancy. As to the experiment for 2), it was clear that the introduction of biotin in the process of in situ PCR method is far effective than to use biotin-labeled primers. It should be noted, however, the introduction of excess labelling by biotin gives poor efficacy in in situ hybrydization. This means the labeling ratio has a key in the successful achievement for the in situ hybrydization histochemistry. The remainders, 3)and 4), have been done with successful results. We will be able to publish the details of this project, when we can cope with occasionally non-reproducible results due to yet unknown reasons.