Detection of messenger RNAs of alpha-fetoprotein and albumin in a human hepatoma cell line by in situ hybridization.
Detection of RNA transcripts within individual cells by in situ hybridization provides a powerful means for identifying specific cell types actively transcribing specific genes. We have applied this technique 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, we found that essentially 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, on the other hand, was below the level of detection by the peroxidase-antiperoxidase method. Consistent with this observation, we could not detect albumin mRNA with 3H-labeled albumin complementary DNA probes. However, 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.
Breborowicz
J
J
Tamaoki
T
T
D000418:Albumins / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000032:analysis*; D002460:Cell Line; D006801:Humans; D007124:Immunoenzyme Techniques; D008113:Liver Neoplasms / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis*; D000509:alpha-Fetoproteins / Q000032:analysis / Q000235:genetics*
D000418:Albumins; D012333:RNA, Messenger; D000509:alpha-Fetoproteins