Single molecule imaging of RNA in situ

Methods Mol Biol. 2011;714:3-13. doi: 10.1007/978-1-61779-005-8_1.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cells, Cultured
  • Fluorescent Dyes / chemistry
  • Image Processing, Computer-Assisted
  • Molecular Imaging / methods*
  • Nucleic Acid Hybridization
  • Oligonucleotide Probes / chemistry
  • Oligonucleotide Probes / genetics
  • Oligonucleotide Probes / isolation & purification
  • RNA / analysis
  • RNA / genetics
  • RNA / metabolism*
  • Rats

Substances

  • Fluorescent Dyes
  • Oligonucleotide Probes
  • RNA