Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors

Abstract

In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid sequences (DNA, RNA) in microscopic preparations of tissues, cells, chromosomes, and linear DNA fibers. To date, a wide variety of research and diagnostic applications of ISH have been described, making the technique an integral part of studies concerning gene mapping, gene expression, RNA processing and transport, the three-dimensional organization of the nucleus, tumor genetics, microbial infections, and prenatal diagnosis. In this review, I first describe the ISH procedure in short and then focus on the currently available non-radioactive probe-labeling and cytochemical detection methodologies that are utilized to visualize one or multiple different nucleic acid targets in situ with different colors. Special emphasis is placed on the procedures applying fluorescence and brightfield microscopy, the simultaneous detection of nucleic acids and proteins by combined ISH and immunocytochemistry, and, in addition, on the recent progress that has been made with the introduction of signal amplification procedures to increase the detection sensitivity of ISH. Finally, a comparison of fluorescence, enzyme cytochemical, and colloidal gold silver probe detection systems will be presented, and possible future directions of in situ nucleic acid detection will be discussed.

High-throughput analysis of gene expression on tissue sections by in situ hybridization

“…The NR-ISH described here is based on dual signal amplification (reviewed in [6,7]) and approaches the sensitivity of radioisotopic ISH as it can detect as few as three transcripts per cell [8]. In the dual amplification strategy, termed ''catalyzed reporter deposition'' (CARD), hapten-tagged RNA (or DNA) probe is hybridized and subsequently detected in situ with an antihapten antibody conjugated to horseradish peroxidase (e.g., POD).…”

Multi-target Chromogenic Whole-mount <em>In Situ</em> Hybridization for Comparing Gene Expression Domains in <em>Drosophila</em> Embryos

“…In recent years significant technological advances in fluorescent ISH (FISH) 2 resulted in dramatically improved sensitivity and resolution, allowing the detection and quantitation of RNA expression at single-cell and sub-cellular levels and RNA visualization up to single molecules 3,4 .While sophisticated single-molecule FISH methods are used for more specialized applications, chromogenic ISH is widespread as a routine RNA in situ detection method in research and clinical diagnostics. For chromogenic detection enzyme precipitation reactions are used, which generate visible products in contrasting colors at the sites of hybridization 5 . This has the advantage that RNA visualization can be combined with routine histological stains and morphological context is immediately evident by standard brightfield microscopy.…”

“…This has the advantage that RNA visualization can be combined with routine histological stains and morphological context is immediately evident by standard brightfield microscopy. Moreover, numerous of the applied color substrates produce precipitates, which are stable in organic and/or aqueous mounting media, so that permanent sample preparations can be obtained 5 .…”

Signal Amplification in Immunohistochemistry at the Light Microscopic Level Using Biotinylated Tyramide and Nanogold-Silver Staining

“…In particular, catalyzed signal amplification (CSA) based on the deposition of labeled tyramide after activation by conversion of H 2 O 2 by peroxidase conjugated with an antibody has greatly increased the sensitivity of IHC and ISH methods (for reviews see Totos et al 1997;Speel 1999;Speel et al 1999). The method was originally developed for ELISA and Western blotting (Bobrow et al 1989(Bobrow et al ,1991(Bobrow et al ,1992 and has been adapted for light microscopy (LM) (Adams 1992;Kerstens et al 1995;Raap et al 1995;Van Gijlswijk et al 1996) and electron microscopy (EM) (Schöfer et al 1997;Bendayan 1997,1999;Punnonen et al 1999).…”

“…We performed this semiquantitative comparative study on rat livers containing colon cancer metastases. Liver parenchyma usually produces higher background levels than other tissues due to high endogenous biotin, avidin, and peroxidase levels (Speel 1999). Therefore, sections containing liver tissue, connective tissue (stroma), and colon cancer (Figure 1) constitute a good model to test the sensitivity and specificity of localization methods.…”