| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1989: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
84 Prostates were obtained at autopsy. Immediately after its removal, each prostate was trimmed into small thin pieces and fixed in acetone. Those were dehydrated with acetone, cleared by methylbenzoate and xylene, and embedded in paraffin. Screening of HE stained sections revealed 22 cases of latent carcinoma. Thick sections were re-cut for subsequent DNA analysis. The weight of 100 micron section ranged from 43 to 56mg, and reduced by 42 % after deparaffinization. DNA was extracted by phenol method, and its amount was 0.13 to 0.15 % of original weight. Agarose gel electrophoresis revealed that the extracted DNA contained fragments longer than ten kilobase.
10 microliters of DNA sample solutions were spotted onto nylon membrane and hybridized with biotin-labeled DNA probe. DNA probes were for c-myc third exon, c-Ha-ras and c-Ki-ras oncogenes. Hybridization temperature was 42 C, and overnight incubation was used. Hybridization buffer contained 5X SSC, 45% formamide. The presence of DNA presence of DNA probe was visualized by alkaline-phosphatase labeled streptavidene/BCIP method. None of the 22 samples showed positive reaction. These results shows that long DNA fragment can be extracted from latent prostatic carcinoma, which is available for analysis of oncogene.
Amplification of c-myc, Ha-ras and Ki-ras oncogenes is not observed in Latent prostatic carcinoma.
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