PUBLICATION

Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos

Authors
Gross-Thebing, T., Paksa, A., Raz, E.
ID
ZDB-PUB-140816-13
Date
2014
Source
BMC Biology   12: 55 (Journal)
Registered Authors
Gross-Thebing, Theresa, Paksa, Azadeh, Raz, Erez
Keywords
none
MeSH Terms
  • Animals
  • Embryo, Nonmammalian/embryology
  • Embryo, Nonmammalian/metabolism
  • Fish Proteins/genetics*
  • Fish Proteins/metabolism
  • Genetic Techniques*
  • In Situ Hybridization*
  • RNA, Messenger/genetics*
  • RNA, Messenger/metabolism
  • Transcription, Genetic*
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish/metabolism
PubMed
25124741 Full text @ BMC Biol.
Abstract

Background

Whole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.

Results

The protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.

Conclusions

By fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.

Errata / Notes
Errata provided by Authors

In Table 2 the composition of the SSCT buffer is provided as-

Reagents
SSCT 0.01%

Composition
0.01% Tween-20, 15 mM NaCl, 1.5 mM TriNaCitratdihydrate, pH 7

This information should be replaced with-

Reagents
0.2X SSCT 0.01%

Composition
0.01% Tween-20, 30 mM NaCl, 3 mM TriNaCitratdihydrate, pH 7

Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping