Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos
By R. Weiszmann, Ann Hammonds, S. Celniker. 2009
102 citations recorded by the source corpus.
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 °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.
Subfields: Genetics, Molecular Biology
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In Genetics
In Molecular Biology
- Erwin Frise
- S. Celniker
- Ann Hammonds
- Manolis Kellis
- Volker Hartenstein
- Niles A. Pierce
- Jason Ernst
- Erik Winfree
Citations in the corpus
- Spatial expression of transcription factors in Drosophila embryonic organ development (2013, methodology): “Methods for in situ hybridization and image capture have been described previously [8,13,22].”
- Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust (2018, background): “, 2008); staining is qualitative rather than quantitative; and spatial resolution is routinely compromised by diffusion of reporter molecules prior to deposition (Tautz and Pfeifle, 1989; Thisse et al., 2004; Acloque et al., 2008; Piette et al., 2008; Thisse and Thisse, 2008; Weiszmann et al., 2009)”
- Systematic image-driven analysis of the spatial Drosophila embryonic expression landscape (2010, methodology): “Using controlled conditions, RNA transcripts were detected by hybridization using an antisense DIG-labeled RNA probe and visualized using immunohistochemistry producing a blue stain (Weiszmann et al , 2009).”