Open AccessNuclear RNA quantification in protoplast cell‐cycle phases
Author(s) -
Bergounioux C.,
Perennes C.,
Gadal P.,
Brown S. C.
Publication year - 1988
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990090113
Subject(s) - protoplast , acridine orange , rna , ribonuclease , dna , biology , microbiology and biotechnology , staining , nuclear dna , cell , cell cycle , biochemistry , genetics , gene , mitochondrial dna
Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.