title: RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH).
abstract: Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule fluorescence in situ hybridization (smFISH)-an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context-provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here, we describe multiplexed error-robust fluorescence in situ hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves.
authors.affiliation: Howard Hughes Medical Institute, Harvard University, Cambridge, MA, United States. Electronic address: lmoffitt@mcb.harvard.edu.
authors.forename: J R
authors.initials: JR
authors.lastname: Moffitt
authors.affiliation: Howard Hughes Medical Institute, Harvard University, Cambridge, MA, United States; Harvard University, Cambridge, MA, United States. Electronic address: zhuang@chemistry.harvard.edu.
authors.forename: X
authors.initials: X
authors.lastname: Zhuang
mesh_terms: D000818:Animals; D001483:Base Sequence; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D012313:RNA / Q000032:analysis*; D059010:Single-Cell Analysis / Q000379:methods; D059467:Transcriptome
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012313:RNA
keywords: In situ hybridization; RNA; Single cells; Single molecules; Single-molecule imaging; Transcriptomics