Journal
NATURE PROTOCOLS
Volume 4, Issue 5, Pages 605-618Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2009.55
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Funding
- NIH [R01 GM076655]
- US Department of Energy [DE-AC0205CH11231]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM076655] Funding Source: NIH RePORTER
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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.
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