Coté B D, Uhlenbeck O C, Steffensen D M
Chromosoma. 1980;80(3):349-67. doi: 10.1007/BF00292689.
The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitative the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate-constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.
将5S和28S核糖体RNA与人成纤维细胞和白细胞进行杂交，以此作为一个模型系统来定量针对人类二倍体细胞类型的原位杂交技术。定量包括在各种杂交程序后，对载玻片上间期细胞核和中期染色体上形成的颗粒数量进行计数（计分）。然后，每张载玻片上每个细胞核的平均颗粒数用于确定杂交百分比。与硝酸纤维素滤膜杂交一样，原位杂交动力学可以用单一的一级速率常数来拟合。然而，原位杂交速率比相应的滤膜杂交速率慢约10倍。对于白细胞和成纤维细胞类型以及中期和间期细胞核，原位杂交效率在5%至15%之间。确定核糖体RNA与二倍体染色体原位杂交反应的参数，确定了将单拷贝基因定位到二倍体染色体所需的实验条件。
