In situ hybridization using synthetic probe.

原位杂交（ISH）是近年来广泛应用于细胞或组织切片中核糖核酸（mRNA）检测和定位的一种方法。为了确定ISH的最佳条件，我们对胎儿肝脏发育过程中克隆的人γ - gtp cDNA进行了Northern blot分析。1) Northern Blot分析早在妊娠12周就可以检测到γ - gtp mRNA，然后逐渐增加到峰值，在12周时约为3倍，在出生前40周。胎儿肝脏mRNA的大小为2.7kb，在人胎儿肾脏和人肝癌以及正常成人肝脏中均检测到锯大小的mRNA。为了确定探针的标记方法，我们进行Northern blot分析，标记随机启动系统和SP6系统。SP6系统的Northern blot分析信号强度优于随机启动系统。3)利用生物素- dutp衍生物转录RNA探针，该衍生物在嘧啶环的5个位置和生物素部分的羧基之间含有一个11个原子的间隔臂。用反义链γ - gtp mRNA原位杂交胎肝细胞，染色胎肝细胞细胞质，并用义链探针进行轻微杂交。需要进一步的研究来消除信号中的非特异性染色，并扩大反应的灵敏度。

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.