title: Quantitative spatial analysis of transcripts in multinucleate cells using single-molecule FISH.
abstract: mRNA positioning in the cell is important for diverse cellular functions and proper development of multicellular organisms. Single-molecule RNA FISH (smFISH) enables quantitative investigation of mRNA localization and abundance at the level of individual molecules in the context of cellular features. Details about spatial mRNA patterning at various times, in different genetic backgrounds, at different developmental stages, and under varied environmental conditions provide invaluable insights into the mechanisms and functions of spatial regulation. Here, we describe detailed methods for performing smFISH along with immunofluorescence for two large, multinucleate cell types: the fungus Ashbya gossypii and cultured mouse myotubes. We also put forward a semi-automated image processing tool that systematically detects mRNAs from smFISH data and statistically analyzes the spatial pattern of mRNAs using a customized MATLAB code. These protocols and image analysis tools can be adapted to a wide variety of transcripts and cell types for systematically and quantitatively analyzing mRNA distribution in three-dimensional space.
authors.affiliation: Howard Hughes Medical Institute, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: ChangHwan
authors.initials: C
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.
authors.forename: Samantha E
authors.initials: SE
authors.lastname: Roberts
authors.affiliation: Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA. Electronic address: amy.gladfelter@dartmouth.edu.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Gladfelter
mesh_terms: D000818:Animals; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry; D005786:Gene Expression Regulation; D015726:Giant Cells / Q000378:metabolism* / Q000648:ultrastructure; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D000072760:Single Molecule Imaging / Q000379:methods / Q000706:statistics & numerical data*; D012984:Software
chemical_list: D005456:Fluorescent Dyes; D012333:RNA, Messenger
keywords: A. gossypii; C2C12 myotubes; Ripley’s H; Single molecule RNA FISH; Spatial analysis