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In situ hybridization using synthetic probe.

使用合成探针进行原位杂交。

基本信息

批准号：
63870002
负责人：
YASUDA Kenjiro
金额：
$ 4.61万
依托单位：
Department of Anatomy, School of Medicine, Keio University Shinanomachi-35, Shinjuku, Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

The in situ hybridization (ISH) has recently become a widely used procedure for detection and localization of nessanger RNA (mRNA) in cells or tissue section. To determine optimal condition for ISH, we performed Northern blot analysis by a cloned cDNA for human gamma-GTP during fetal development of liver.1) Northern Blot Analysis gamma-GTP mRNA could be detected as early as at 12 weeks of gestation, and then increased gradually to a peak, approximately threefold of the amount at 12th week, at 40th week just before birth. The size of mRNA in fetal liver was 2.7kb and the mRNA of the saw size were detected both in human fetal kidney and human hepatocellular carcinoma as well as normal adult liver.2) Probe Labeling To determine the method of probe labeling, we performed Northern blot analysis, with labeling of random priming system and SP6 system. The signal intensity of Northern blot analysis with SP6 system was superior to that with random priming system.3) In Site Hybridization RNA probe were transcripted using biotinyl-dUTP derivative that contained an 11-atom spacer arm between the 5 position of the pyrimidine ring and the carboxyl group of the biotin moiety. When fetal liver were hybridized in situ with anti-sense strand gamma-GTP mRNA, the cytoplasm of the fetal hepatocytes were stained, and they were slightly hybridized with sense strand probe. Further study are needed to eliminate non specific staining from signals and to amplify the sensitivity of the reaction.

原位杂交(ISH)是近年来被广泛应用于细胞或组织切片中核糖核酸(nessanger RNA，mRNA)检测和定位的方法。为了确定最佳的ISH条件，我们利用克隆的人肝脏发育过程中的人γ-GTP的cDNA进行了Northern印迹分析。1)Northern Blot分析表明，伽马-GTP的mRNA最早在12周时就可以检测到，然后逐渐增加到12周时的峰值，大约是出生前40周时的3倍。在人胎肝、人胎肾、人肝细胞癌和正常成人肝组织中均检测到SAW大小的mRNA。2)为了确定探针标记的方法，我们进行了Northern杂交分析，分别用随机引物法和SP6系统进行标记。3)原位杂交RNA探针的转录使用生物素dUTP衍生物，其5位与生物素部分的羧基之间含有11个原子的间隔臂。当胎肝与反义链γ-GTP mRNA原位杂交时，胎肝细胞胞浆染色，与正义链探针轻度杂交。需要进一步的研究来消除信号中的非特异性染色，并放大反应的敏感性。