Fig. S2. Detection of RNA in fixed cells. (A) For detection of the RNA at the transcription site transcriptioanlly induced cells were fixed and the hybridized with 2 different probes to the transcribed RNA. An activated gene locus (30 min post-induction) detected by the YFP-lac repressor protein, (B) colocalized with the RNA as seen by FISH using a Cy3-probe to the exon sequence and a (C) Cy5-probe to the MS2 repeats, (D) merge. Bar = 0.5 µm. Sensitive RNA quantification: (E) Example of dilution curve obtained for the Cy3-exon5 probe. Total fluorescent intensities (TFI) were collected for serial dilutions of the probe and plotted giving rise to a straight line. The slope of this line is the TFI per one probe molecule. (F) One plane from a 3D stack of a nucleus (cells activated for 30 min) analyzed for RNA molecule quantification with a YFP-lac repressor protein tagged locus, (G) the Cy3-exon5 RNA signal in the same plane (before deconvolution), (H) a color map of the total RNA molecules per spot detected after deconvolution. RNA molecules detected: white = 1 molecule, green = 2 molecules, orange = 3 molecules. Blue = transcription site (arrow). Bar = 2 µm. (I) Distribution of the average number of nuclear RNA molecules per particle detected over a 2 hour induction (analysis of 6707 nuclear transcripts from 21 doxycycline-induced cells) showed that the majority (~70%) of transcripts hybridized with a single probe indicating that these nuclear particles represent single RNA transcripts. A slight decrease (P<0.05) in the fraction of single transcripts was observed over time post-induction (76% at 10 min to 64% at 2 hrs).