In situ hybridization using synthetic probe.

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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.