Single-molecule mRNA detection and counting in mammalian tissue.
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.
Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Anna
A
Lyubimova
Shalev
S
Itzkovitz
Jan Philipp
JP
Junker
Zi Peng
ZP
Fan
Xuebing
X
Wu
Alexander
A
van Oudenaarden
D000818:Animals; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007421:Intestine, Small / Q000378:metabolism; D051379:Mice; D015345:Oligonucleotide Probes; D063150:Polycomb Repressive Complex 1 / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D012984:Software; D014584:User-Computer Interface
C489421:Bmi1 protein, mouse; C493597:Lgr5 protein, mouse; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D063150:Polycomb Repressive Complex 1