Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes

Methods Enzymol. 2010;472:365-86. doi: 10.1016/S0076-6879(10)72004-8.

Abstract

Measurements of gene expression within single cells have revealed startling variability otherwise hidden in bulk measurements. Here, we present an in situ hybridization method capable of detecting individual mRNA molecules, thus permitting the accurate quantification and localization of mRNA within fixed sample. Our in situ protocol involves probing the target mRNA using a series of singly labeled oligonucleotide probes. This method is simple to implement and is applicable to a variety of biological samples. We also discuss some aspects of image processing required for analyzing the resulting data.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid
  • Humans
  • Image Processing, Computer-Assisted / methods
  • In Situ Hybridization / methods*
  • Microscopy, Fluorescence / methods
  • Nucleic Acid Hybridization / methods
  • Oligonucleotide Probes / chemistry*
  • Oligonucleotide Probes / genetics
  • RNA / chemistry*
  • RNA / genetics
  • Yeasts / genetics

Substances

  • Oligonucleotide Probes
  • RNA