Deciphering Transcriptional Dynamics In Vivo by Counting Nascent RNA Molecules
Fig 1
Experimental methods to count nascent RNA (transcribing RNA polymerase molecules).
(A) Electron micrographs of intact chromosomes extracted from cells provide images of transcribing polymerases along a gene (also referred to as Miller spreads). This method allows one to count the number of RNAP molecules that are actively transcribing a gene of interest across a population of genetically identical cells. Histograms for the distribution of number of Pol I molecules is shown along rDNA, for a wild type yeast cell (adapted from [67]). (B) Fluorescence in Situ Hybridization (FISH) in single cells provide the intensity of the transcription site, which can then be used to count the number of nascent RNAs for a particular gene. Histogram for the nascent RNA distribution is shown for MDN1 gene in yeast (adapted from [25]) (C) Such measurements can be used to count the number of nascent RNA transcripts using the fact that the length of nascent RNA transcripts are shorter than the mRNA transcripts. Histograms for mRNA distribution [68] in ES cells is shown.