Sequential RNA and DNA fluorescence in situ hybridization (Prot 39)
An increasing body of evidence indicates that the spatial positioning of genes in the interphase nucleus is highly relevant for their function (Lanctot et al, 2007; Meaburn & Misteli, 2007; Misteli, 2007). Fluorescence in situ hybridization (FISH) is a powerful technique to map gene loci in the interphase nucleus. Depending on protocol FISH can either detect DNA or RNA. Both methods have limitations. DNA FISH only detects the physical location of a gene, but can not detect gene activity. RNA FISH, on the other hand, detects transcripts, but might miss a significant number of alleles, since not all alleles of a gene are necessarily transcribed simultaneously. The most efficient way to map gene loci and their activity is sequential RNA and DNA FISH. This is an important technique to uncover how gene positioning is linked to activity. [...]
Cell Biology of Genomes, National Cancer Institute, NIH - 41 Library Drive, Bldg. 41 - Bethesda, MD 20892, USA
Live imaging with Drosophila tissue culture cells (Prot 25)
Live imaging provides an important complementation to the "snapshot" view obtained in fixed tissue by immunofluorescence. It allows following dynamic cellular processes as they unfold "in vivo", and often reveals a degree of complexity impossible to study in still images. Thanks to the increasing interest in this technique, significant technical improvements have been made in the recent years in microscopy leading to more sensitive and faster cameras, more efficient filter sets and a variety of different microscope systems to choose from. The temporal dimension of cellular processes in microscopy is still often neglected due to the difficulties in technical setup and the necessity to fabricate your own tools. Now the majority of the required equipment is commercially available from microscope manufacturers and related companies including environmental chambers, CO2-control and temperature control.
Often used in conjunction with time-lapse microscopy are fluorescent proteins, such as the Green Fluorescent Protein (GFP) or the Red Fluorescent Protein (RFP; Shaner et al., 2004; Shaner et al., 2005; Wiedenmann et al., 2004) and live dyes. Likewise this is a rapidly developing field of research, which has provided a variety of different "flavors" of fluorescent proteins for the scientific community, with properties specifically suited for different live-imaging applications (for a guide to choose the right one, see Shaner et al., 2005).
MPI of Immunobiology
Freiburg 79108, Germany
RNA FISH on cultured cells in interphase (Prot 6)
Fluorescence in situ hybridization (FISH) has become a widely used method in genome and molecular genetic studies. The technique is highly versatile and has been adapted to carry out genome-wide screenings, microarray quantifications, cancer cytogenetics analysis, and RNA expression and localization studies. The study of intracellular RNA localization using RNA FISH provides insights into the in situ physical characteristics of transcription and intracellular RNA transport in individual cells. In our lab, we use RNA FISH to detect the localization of Xist RNA, a nuclear non-coding transcript that coats the entire chromosome from which it is transcribed.
The RNA FISH technique requires the generation of a labeled probe, hybridization of the probe to a fixed sample, and subsequently, detection of the labeled probe using microscopy.
Research Institute of Molecular Pathology (IMP)
Dr. Bohr-Gasse 7
A1030 Vienna, Austria
Multicolour 3D-FISH in vertebrate cells (Prot 23)
Multicolour 3D-FISH in combination with confocal microscopy, 3D image reconstruction and quantitative image analysis is an efficient tool for the analysis of the 3D genome structure and of the spatial relationship of defined nuclear targets comprising entire chromosome territories down to the level of single gene loci. Until a few years ago the drawback of confocal microscopy was its limitation to three or at maximum to four different fluorochromes that could be visualized simultaneously. Recent developments of a "new generation" of confocal microscopes allow the simultaneous excitation and distinct visualization of five different fluorochromes (the number can be increased if colour unmixing software is used) within one experiment, opening the way for a simultaneous delineation of numerous differently labeled intranuclear targets. [...]
AG Thomas Cremer
Department Biology II
Grosshadernerstr. 2 - 82152 Martinsried-Planegg, Germany
Two-colour fluorescent in situ DNA hybridization on whole mount Drosophila embryos and larval imaginal discs (Prot 7)
Increasing evidence in literature reveals that the organization of chromosomal domains in the cell nucleus plays an important role in gene expression during cellular differentiation and development (Spector, 2003, Taddei et al, 2004). Fluorescent in situ DNA hybridization (FISH) allows to study the positioning of single copy genes into the nucleus, and is therefore widely applied to address the question of how is gene positioning regulated.
We describe here a two-colour FISH method for interphase nuclei of whole mount drosophila embryos. This procedure has been adapted from the protocol described by Gemkow et al, 1998, and allows us to study the relative position of two distinct single copy genes in the nucleus. This FISH protocol can be applied to other drosophila tissues, such as larval imaginal discs (Bantignies et al, 2003). For simplicity, we will use "tissues" throughout the text to design embryos and larval discs in the steps common to all tissues, whereas we will specify "embryos" or "larval imaginal discs" in those steps that apply to only one type of tissue.
Institute of Human Genetics
CNRS UPR 1142 - 141, rue de la Cardonille - 34396 Montpellier cedex 5, France