In situ hybridization using synthetic probe.

原位杂交技术是近年来发展起来的一种检测和定位细胞或组织切片中nessanger RNA的方法。为了确定原位杂交的最佳条件，我们用克隆的人胎儿肝脏发育过程中γ-GTP的cDNA进行了北方印迹分析。1）北方印迹分析γ-GTP mRNA早在妊娠12周就可检测到，然后逐渐增加，到12周时达到峰值，约为出生前40周的3倍。2）探针标记为确定探针的标记方法，我们采用随机引物系统和SP6系统进行了北方印迹分析，并对探针的标记方法进行了比较。SP6系统的Northern杂交信号强度优于随机引物系统。3）原位杂交RNA探针的转录是以生物素-dUTP衍生物为探针，该衍生物在嘧啶环的5位和生物素的羧基之间含有一个11个原子的间隔臂。胎肝与反义链γ-GTP mRNA原位杂交时，胎肝细胞胞浆染色，与正义链探针有轻度杂交。需要进一步的研究来消除信号中的非特异性染色，并提高反应的灵敏度。

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.