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 °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.
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Acknowledgements
We thank current and former members of the BDGP, especially Amy Beaton, Joe Carlson, Erwin Frise, Elaine Kwan, Todd Laverty, Barret Pfeiffer, Stephen Richards, Mark Stapleton, Pavel Tomancak and Gerry Rubin for their contributions to these protocols as part of the systematic determination of patterns of gene expression in the Drosophila embryogenesis project. We thank Ken Wan for critical comments on the manuscript. This work was supported by the NIH Grant R01 GM076655 (SEC) through the US Department of Energy under contract no. DE-AC02-05CH11231.
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Weiszmann, R., Hammonds, A. & Celniker, S. Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos. Nat Protoc 4, 605–618 (2009). https://doi.org/10.1038/nprot.2009.55
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DOI: https://doi.org/10.1038/nprot.2009.55
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