Cells were transfected and grown for 24 h, minichromosomal transcripts were detected by RNA FISH, DNA was counterstained with DAPI, and images were collected
(A) Two sets of four views of one field are shown. (bottom) Percentages (± SD) of green foci that overlap red foci, and vice versa. (i–iv) Transfection with II,,pA. An untransfected cell (arrowhead) contains no or transcripts. The other contains many cytoplasmic transcripts but few transcripts; its nucleus contains some red and green foci (marking nascent RNA at transcription sites) against a general background (marking transcripts on their way to the cytoplasm). (insets) Two foci with both red and green fluorescence; this is expected, as the plasmid encodes both and (on the backbone). (v–viii) Cotransfection with II,,pA and II,,pA, which differ solely in coding region. The two central (transfected) cells contain and RNA mainly in the cytoplasm, with some in the nuclear foci. (insets) Nuclear focus with both types of RNA. Insets show an enlarged view of the boxed portions. (B) Discriminating between nuclear foci and background. Intensities are expressed relative to those given by fluorescent reference beads, and the fraction of foci in 200 cells with relative intensities of 0–0.05, 0.06–0.1, etc., is indicated. Promoterless 0,,pA gives faint signal due to autofluorescence (equivalent to that seen in mock-transfected cells, not depicted; gray bar) and read-through from the SV40 early promoter into (red bars). For plasmids with promoters (e.g., II,,pA), only foci with intensities greater than the maximum read-through (green dotted line) were considered. (C) Cells were transfected with II,,pA and incubated with or without α-amanitin and RNase, DNA was stained with DAPI, and transcripts were detected; the treatments abolish signal. Three sets of two views of one field are shown. Bars: (A) 5 μm; (C) 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Similar active genes cluster in specialized transcription factories"</p><p></p><p>The Journal of Cell Biology 2008;181(4):615-623.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386102.</p><p></p
