High-Throughput RNA In Situ Hybridization in Mouse Retina

The Salomon H. Snyder Department of Neuroscience, John Hopkins University School of Medicine, Baltimore, MD, USA, .
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2013; 935:215-226. DOI: 10.1007/978-1-62703-080-9_15
Source: PubMed


The introduction of large-scale gene expression profiling studies has greatly increased the need to rapidly obtain high-quality cellular expression patterns of genes found to exhibit differential expression. The use of large-scale nonradioactive RNA in situ hybridization makes this possible, and greatly increases the general usefulness of this data. Here, we describe protocols for parallel analysis of up to 50 different gene-specific probes in mouse retinal sections.

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    ABSTRACT: In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)-RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase-conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform.
    No preview · Article · Nov 2013 · Nature Protocol