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Single-molecule mRNA detection and counting in mammalian tissue

Item Type:Article
Title:Single-molecule mRNA detection and counting in mammalian tissue
Creators Name:Lyubimova, A. and Itzkovitz, S. and Junker, J.P. and Fan, Z.P. and Wu, X. and van Oudenaarden, A.
Abstract:We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNA dye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3-7 d, depending on the type of analysis.
Keywords:Fluorescence In Situ Hybridization, Green Fluorescent Proteins, G-Protein-Coupled Receptors, Messenger RNA, Oligonucleotide Probes, Polycomb Repressive Complex 1, Proto-Oncogene Proteins, Small Intestine, Software, User-Computer Interface, Animals, Mice
Source:Nature Protocols
Publisher:Nature Publishing Group
Page Range:1743-1758
Date:September 2013
Official Publication:https://doi.org/10.1038/nprot.2013.109
PubMed:View item in PubMed

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