title: Rapid Ovary Mass-Isolation (ROMi) to Obtain Large Quantities of Drosophila Egg Chambers for Fluorescent In Situ Hybridization.
abstract: Isolation of large quantities of tissue from organisms is essential for many techniques such as genome-wide screens and biochemistry. However, obtaining large quantities of tissues or cells is often the rate-limiting step when working in vivo. Here, we present a rapid method that allows the isolation of intact, single egg chambers at various developmental stages from ovaries of adult female Drosophila flies. The isolated egg chambers are amenable for a variety of procedures such as fluorescent in situ hybridization, RNA isolation, extract preparation, or immunostaining. Isolation of egg chambers from adult flies can be completed in 5 min and results, depending on the input amount of flies, in several milliliters of material. The isolated egg chambers are then further processed depending on the exact requirements of the subsequent application. We describe high-throughput in situ hybridization in 96-well plates as example application for the mass-isolated egg chambers.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany. jambor@mpi-cbg.de.
authors.forename: Helena
authors.initials: H
authors.lastname: Jambor
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany.
authors.forename: Pavel
authors.initials: P
authors.lastname: Mejstrik
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307, Dresden, Germany. tomancak@mpi-cbg.de.
authors.forename: Pavel
authors.initials: P
authors.lastname: Tomancak
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods; D004331:Drosophila melanogaster / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure*; D004625:Embryo, Nonmammalian / Q000378:metabolism / Q000648:ultrastructure*; D005260:Female; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009866:Oogenesis / Q000235:genetics; D061848:Optical Imaging / Q000379:methods; D010053:Ovary / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure*; D015053:Zygote / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure*
keywords: Drosophila; Egg chamber; FISH; Mass isolation; Ovary; ROMi; Rapid ovary mass-isolation