DETECTION OF MESSENGER-RNAS OF ALPHA-FETOPROTEIN AND ALBUMIN IN A HUMAN HEPATOMA-CELL LINE BY INSITU HYBRIDIZATION

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  • 1 January 1985
    • journal article
    • research article
    • Vol. 45  (4) , 1730-1736
Abstract
Detection of RNA transcripts within individual cells by in situ hybridization provides a powerful means for identifying specific cell types actively transcribing specific genes. This technique was applied in order to analyze expression of the .alpha.-fetoprotein and albumin genes in a human hepatoma cell line, HuH-7. Using either 3H- or 35S-labeled human .alpha.-fetoprotein complementary DNA clone as a probe, all HuH-7 cells contained .alpha.-fetoprotein mRNA, although in various amounts. This correlated well with the presence of .alpha.-fetoprotein in all cells as detected by the peroxidase antiperoxidase immunoenzyme method. The intracellular concentration of albumin was below the level of detection by the peroxidase-antiperoxidase method. Consistent with this observation, albumin mRNA could not be detected with 3H-labeled albumin complementary DNA probes. The use of 35S-labeled probes having higher specific activities and higher efficiency of grain development resulted in the detection of albumin mRNA in a small percentage of HuH-7 cells. A variety of parameters involved in the in situ hybridization technique was examined to establish conditions suitable for demonstrating the presence of high- and low-copy numbers of mRNA in various cell and tissue preparations.

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