A solution hybridization/RNase protection assay with riboprobes to determine absolute levels of apoB, A-I, and E mRNA in human hepatoma cell lines.
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.
Azrolan
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Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, NY 10021.
Breslow
J L
JL
D016632:Apolipoprotein A-I; D001053:Apolipoproteins / Q000235:genetics*; D001054:Apolipoproteins A / Q000032:analysis; D001055:Apolipoproteins B / Q000032:analysis; D001057:Apolipoproteins E / Q000032:analysis; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis; D012260:Ribonucleases; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D001054:Apolipoproteins A; D001055:Apolipoproteins B; D001057:Apolipoproteins E; D015347:RNA Probes; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012260:Ribonucleases