An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs

Hui Yang, Ina B. Wanner, Stephen D. Roper, Nirupa Chaudhari

Research output: Contribution to journalArticle

153 Scopus citations

Abstract

In situ hybridization (ISH) using nonradioactive probes enables mRNAs to be detected with improved cell resolution but compromised sensitivity compared to ISH with radiolabeled probes. To detect rare mRNAs, we optimized several parameters for ISH using digoxygenin (DIG)-labeled probes, and adapted tyramide signal amplification (TSA) in combination with alkaline phosphatase (AP)-based visualization. This method, which we term TSA-AP, achieves the high sensitivity normally associated with radioactive probes but with the cell resolution of chromogenic ISH. Unlike published protocols, long RNA probes (up to 2.61 KB) readily permeated cryosections and yielded stronger hybridization signals than hydrolyzed probes of equivalent complexity. RNase digestion after hybridization was unnecessary and led to a substantial loss of signal intensity without significantly reducing nonspecific background. Probe concentration was also a key parameter for improving signal-to-noise ratio in ISH. Using these optimized methods on rat taste tissue, we detected mRNA for mGluR4, a receptor, and transducin, a G- protein, both of which are expressed at very low abundance and are believed to be involved in chemosensory transduction. Because the effect of the tested parameters was similar for ISH on sections of brain and tongue, we believe that these methodological improvements for detecting rare mRNAs may be broadly applicable to other tissues.

Original languageEnglish (US)
Pages (from-to)431-445
Number of pages15
JournalJournal of Histochemistry and Cytochemistry
Volume47
Issue number4
DOIs
StatePublished - Apr 1999

Keywords

  • In situ hybridization
  • Rare mRNA
  • Sensory transduction
  • Taste
  • Tyramide amplification

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Fingerprint Dive into the research topics of 'An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs'. Together they form a unique fingerprint.

  • Cite this