Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.
We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.
Tang
F J
FJ
Division of Biophysics, School of Hygiene, Johns Hopkins University, Baltimore, Maryland 21205.
Ts'o
P O
PO
Lesko
S A
SA
D002453:Cell Cycle; D002460:Cell Line; D003592:Cytophotometry; D004247:DNA / Q000032:analysis*; D005354:Fibrosarcoma / Q000473:pathology*; D005786:Gene Expression Regulation; D006801:Humans; D007370:Interferon Type I / Q000032:analysis* / Q000235:genetics; D009693:Nucleic Acid Hybridization; D011003:Ploidies; D012333:RNA, Messenger / Q000032:analysis*
D007370:Interferon Type I; D012333:RNA, Messenger; D004247:DNA