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Single Molecule Imaging of RNA In Situ

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

Abstract

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 

Notes

Acknowledgements

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.

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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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