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.
Berkeley Drosophila Genome Project, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Richard
R
Weiszmann
Ann S
AS
Hammonds
Susan E
SE
Celniker
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