Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos.
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.
Weiszmann
Richard
R
Berkeley Drosophila Genome Project, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Hammonds
Ann S
AS
Celniker
Susan E
SE
D000818:Animals; D003001:Cloning, Molecular; D004330:Drosophila / Q000196:embryology / Q000235:genetics*; D046149:Embryo Culture Techniques; D004625:Embryo, Nonmammalian; D047108:Embryonic Development / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis*; D015347:RNA Probes
D015347:RNA Probes; D012313:RNA