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

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

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  • 1Berkeley Drosophila Genome Project, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

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.

PMID:
19360017
[PubMed - indexed for MEDLINE]
PMCID:
PMC2780369
Free PMC Article

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