Whole mount RNA fluorescent in situ hybridization of Drosophila embryos

Félix Legendre, Neal Cody, Carole Iampietro, Julie Bergalet, Fabio Alexis Lefebvre, Gaël Moquin-Beaudry, Olivia Zhang, Xiaofeng Wang, Eric Lécuyer

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Original languageEnglish
Pages (from-to)e50057
JournalJournal of Visualized Experiments
Issue number71
DOIs
StatePublished - 30 Jan 2013
Externally publishedYes

Keywords

  • Animal model
  • Cellular biology
  • Developmental biology
  • Drosophila
  • Embryo
  • Embryogenesis
  • FISH
  • Fluorescent in situ hybridization
  • Fruit fly
  • Gene expression pattern
  • Genetics
  • Genomics
  • Issue 71
  • Knockout
  • Molecular biology
  • RNA
  • RNA localization
  • TSA
  • Tyramide signal amplification
  • Whole mount

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