Single Molecule Imaging of RNA In Situ

  • Mona Batish
  • Arjun Raj
  • Sanjay TyagiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 714)


This protocol describes a method to image individual mRNA molecules in situ. About 50 oligonucleotides complementary to different regions of a target mRNA species are used simultaneously. Each probe is labeled with a single fluorescent moiety. When these probes bind to their target, each mRNA molecule becomes so intensely fluorescent that it can be seen as a fine fluorescent spot. Several different mRNA species can be detected in multiplex imaging using differently colored probe sets for each species. An automated image-processing program is used to count the number of mRNA molecules of each species that are expressed in each cell, thus yielding single-cell gene expression profiles.

Key words

mRNA visualization FISH Single molecule detection In situ hybridization MATLAB image processing Gene expression profiling 



This work was supported by a grant from the National Institutes of Mental Health (MH079197). Authors acknowledge help from Salvatore A.E. Marras in probe labeling and purification.


  1. Femino, A.M., Fay, F.S., Fogarty, K. and Singer, R.H. (1998) Visualization of single RNA transcripts in situ. Science, 280, 585–590.PubMedCrossRefGoogle Scholar
  2. Lu, J. and Tsourkas, A. (2009) Imaging individual microRNAs in single mammalian cells in situ. Nucleic Acids Res, 37, e100.PubMedCrossRefGoogle Scholar
  3. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. and Tyagi, S. (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods, 5, 877–879.PubMedCrossRefGoogle Scholar
  4. Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y. and Tyagi, S. (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol, 4, e309.PubMedCrossRefGoogle Scholar
  5. Raj, A., Rifkin, S.A., Andersen, E. and van Oudenaarden, A. (2010) Variability in gene expression underlies incomplete penetrance. Nature, 463, 913–918.PubMedCrossRefGoogle Scholar
  6. Banker, G. and Goslin, K. (ed.) (1998) Culturing Nereve Cells. MIT Press, Cambridge, MA.Google Scholar
  7. Yildiz, A., Forkey, J.N., McKinney, S.A., Ha, T., Goldman, Y.E. and Selvin, P.R. (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science, 300, 2061–2065.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Public Health Research Institute Center, New Jersey Medical SchoolUniversity of Medicine and Dentistry of New JerseyNewarkUSA

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