title: Super-resolution measurement of distance between transcription sites using RNA FISH with intronic probes.
abstract: Nascent transcripts being copied from specific human genes can be detected using RNA FISH (fluorescence in situ hybridization) with intronic probes, and the distance between two different nascent transcripts is often measured when studying structure-function relationships. Such distance measurements are limited by the resolution of the light microscope. Here we describe methods for measuring these distances in cultured cells with a precision of a few tens of nanometers, using equipment found in most laboratories (i.e., a wide-field fluorescence microscope equipped with a charged-coupled-device camera). Using images of pairs of transcripts that are often co-transcribed, we discuss how selection of cell type, design of FISH probes, image acquisition, and image processing affect the precision that can be achieved.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Larkin
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address: peter.cook@path.ox.ac.uk.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Cook
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007438:Introns; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods; D012984:Software; D016707:Tissue Fixation / Q000379:methods; D014158:Transcription, Genetic
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes; D012333:RNA, Messenger; D012097:Repressor Proteins; C507993:SAMD4A protein, human
keywords: Colocalization; Diffraction limit; Inner-nuclear distance; Long gene; Nascent intronic RNA FISH; Super-resolution localization; Transcription location