Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos

Nat Protoc. 2009;4(5):605-18. doi: 10.1038/nprot.2009.55.

Abstract

We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4 degrees C for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

Publication types

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

MeSH terms

  • Animals
  • Cloning, Molecular
  • Drosophila / embryology
  • Drosophila / genetics*
  • Embryo Culture Techniques
  • Embryo, Nonmammalian
  • Embryonic Development / genetics*
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation, Developmental
  • In Situ Hybridization / methods*
  • Polymerase Chain Reaction
  • RNA / analysis*
  • RNA Probes

Substances

  • RNA Probes
  • RNA