Azrolan N, Breslow J L
Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, NY 10021.
J Lipid Res. 1990 Jun;31(6):1141-6.
A solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.
我们开发了一种使用核糖探针的溶液杂交/RNase保护分析方法来定量载脂蛋白mRNA的浓度。以前，放射性标记的DNA探针已用于溶液杂交/S1核酸酶保护分析以达到此目的。与以前的分析相比，新的分析方法在探针制备和杂交方面所需时间更少。此外，用于制备核糖探针的载体还可方便地用于产生生成标准曲线所需的cRNA，以定量绝对载脂蛋白mRNA水平。溶液杂交RNase保护分析用于定量四种人肝癌细胞系HepG2、Hep3B、WRL-68、SK-Hep2中apoB、A-I和E mRNA的水平。HepG2和Hep3B细胞，而非WRL-68和SK-Hep2细胞，所有三种载脂蛋白mRNA的浓度与体内肝脏相当。这些数据表明，HepG2和Hep3B是研究肝脏特异性载脂蛋白基因表达的合适模型。
