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

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. Accepted: 9 June 1999     

In situ hybridization in human genome mapping

The methodological possibilities of non-radioactive in situ hybridization of nucleic acids and its application for an immediate localization of genes and chromosomes are discussed. The advantages of a non-isotope probe labeling versus a use of radioactive substances are emphasized. Various types of compounds used as a label are distinguished. The principles of use of the above labels and the ways to improve the method in terms of increasing its sensitivity are considered. Some results obtained while using non-radioactive labelled probes are reported. It is shown promising to use this method for molecular-genetic analysis of DNA polymorphism in human genome.     

Non-isotopic RNA probes. Comparison between different labels and detection systems

Several studies have shown the use of non-radioactive labelled DNA probes for in situ hybridisation, mainly to identify cellular DNA. In this study mRNA in situ hybridisation was performed on rat pituitary with biotinylated complementary (c) RNA probes for rat prolactin and growth hormone (GH), and compared with radioactive 35S-radiolabelled probes. Biotinylated cRNA probes were labelled with either biotin-11-UTP or with allylamine-UTP, the latter method being able to produce a higher yield of labelled RNA. Different detection systems were tested, and hybridisation signal was seen in cells of anterior pituitary with both types of biotinylated probes. The signals were detected using either avidin-biotin-complex with peroxidase (ABC), peroxidase-anti-peroxidase (PAP) or gold-silver methods. ABC peroxidase detected using glucose oxidase-diaminobenzidine (DAB)-nickel solution appeared to be the best method for detecting labelled RNA probes, with very strong signal and low background. The biotinylated probes were comparable in sensitivity to the radiolabelled probes in detecting prolactin and GH mRNAs in the anterior lobe of the rat pituitary. These results indicate an alternative methods of labelling and detection of biotinylated probes which could have a potential role in research and diagnostic techniques.     

Non-isotopic RNA probes

Summary Several studies have shown the use of non-radioactive labelled DNA probes for in situ hybridisation, mainly to identify cellular DNA. In this study mRNA in situ hybridisation was performed on rat pituitary with biotinylated complementary (c) RNA probes for rat prolactin and growth hormone (GH), and compared with radioactive ³⁵S-radiolabelled probes. Biotinylated cRNA probes were labelled with either biotin-11-UTP or with allylamine-UTP, the latter method being able to produce a higher yield of labelled RNA. Different detection systems were tested, and hybridisation signal was seen in cells of anterior pituitary with both types of biotinylated probes. The signals were detected using either avidin-biotin-complex with peroxidase (ABC), peroxidase-anti-peroxidase (PAP) or gold-silver methods. ABC peroxidase detected using glucose oxidase-diaminobenzidine (DAB)-nickel solution appeared to be the best method for detecting labelled RNA probes, with very strong signal and low background. The biotinylated probes were comparable in sensitivity to the radiolabelled probes in detecting prolactin and GH mRNAs in the anterior lobe of the rat pituitary. These results indicate an alternative methods of labelling and detection of biotinylated probes which could have a potential role in research and diagnostic techniques.     

Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction

The use of in situ techniques to detect DNA and RNA sequences has proven to be an invaluable technique with paraffin-embedded tissue. Advances in non-radioactive detection systems have further made these procedures shorter and safer. We report the detection of Trypanosoma cruzi, the causative agent of Chagas disease, via indirect and direct in situ polymerace chain reaction within paraffin-embedded murine cardiac tissue sections. The presence of three T. cruzi specific DNA sequences were evaluated: a 122 base pair (bp) sequence localized within the minicircle network, a 188 bp satellite nuclear repetitive sequence and a 177 bp sequence that codes for a flagellar protein. In situ hybridization alone was sensitive enough to detect all three T. cruzi specific DNA sequences.     

Hybridocytochemistry with non-radioactive probes as a tool for biomedical research and cytodiagnostics

The advent of DNA and RNA labelling techniques which allow non-autoradiographic detection of specific nucleic acid sequences has opened new possibilities for rapid diagnosis at the nucleic acid level. There are already practical applications. However, in order to fully compete with radioactive procedures an increase in sensitivity is still desired.     

A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback

We have developed a non-radioactive in situ hybridization technique for the localization of RNA in whole mount Drosophila embryos. After fixation, whole embryos are hybridized in situ with a DNA probe which has been labeled with digoxygenin. The hybridization products are detected by using a phosphatase-coupled antibody against digoxygenin. In parallel experiments, embryos can be treated with an antibody directed against the corresponding protein product to allow the detection of its distribution using standard immunochemical techniques. We have used this approach to compare the spatial and temporal distribution patterns of the RNA and protein products of the segmentation gene hunchback (hb) during the early stages of embryogenesis. This comparison revealed translational control of the maternally derived hb mRNA, which was difficult to detect by conventional techniques. The non-radioactive in situ hybridization method is as sensitive as conventional methods, but is faster and easier to perform. This may make it a useful tool for a variety of other systems.     

A Comparison of Digoxigenin and Biotin Labelled DNA and RNA Probes for in Situ Hybridization

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes acheived maximal sensitivity without use of biotin-streptavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.     

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

Summary The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.     

Enzymatic labeling of nucleic acids

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.     

Non-radioactive detection of single-copy DNA-DNA hybrids

A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-radioactive labeling and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase. The main advantages of this procedure are the possibility to reuse the blots easily and the much shorter detection time compared to radioactive detection methods.     

New prospects for the diagnosis of viral infections

The diagnosis of viral infections is important for the accurate management of patients with infectious diseases and for the monitoring of the course of epidemics in susceptible populations. The utility of traditional viral diagnostic assays is limited by the time, expense, and expertise required for the performance of tissue culture techniques. Similarly, the application of immunoassay techniques has been inhibited by the limited degrees of sensitivity and specificity which can be attained by most immunoassay methods. Recently, techniques for the identification of DNA and RNA have been applied to the detection of viral nucleic acids in clinical samples. Such assays have a number of potential advantages over corresponding immunoassays directed at the detection of viral antigens. In order to be generally applicable to clinical diagnosis, however, formats for the detection of viral nucleic acids have to be devised which allow for the reproducible quantitation of target DNA or RNA in human body fluids. Furthermore, formats need to be devised which allow enhanced assay sensitivity while maintaining high degrees of specificity and reproducibility. The use of non-isotopic labeling, liquid-phase hybridization, and target amplification techniques offers partial solutions to these problems. The development of practical assays for the detection of viral nucleic acids under a broad range of clinical and laboratory conditions would represent a major advance in the ability of physicians to care for patients with suspected infections.     

A non-radioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands.

Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The principle of the method is based on the reaction of the mercurated pyrimidine residues of the in situ hybridized probe with the sulfhydryl group of a ligand which contains a hapten. Next, the hapten is immunocytochemically detected. Previous experiments showed that stable coupling of the sulfhydryl ligands could only be obtained when positively charged amino groups are present in the ligand. On basis of this finding, ligands were synthesized containing a sulfhydryl group, two lysyl residues and hapten groups such as trinitrophenyl, fluorescyl and biotinyl. The ligands, free or bound to mercurated nucleic acids, were immunochemically characterized in ELISAs. The method was shown to be specific and sensitive in the detection of target DNA in situ on microscopic preparations and in dot-blot hybridization reactions on nitrocellulose.     

Immunochemical approaches to gene probe assays

Antibodies specific for helical nucleic acids can be applied to assays for hybridized DNA and/or RNA. The assays can use either radioactive or nonradioactive detection systems. Antibodies specific for RNA-DNA hybrids are applicable to assays for measuring hybrid helices that are immobilized on plastic or nitrocellulose, whether the helices are preformed in solution or are formed on the solid-phase support. Alternatively, anti-RNA-DNA hybrid antibodies can be immobilized and used to capture hybrids formed in solution, resulting in an assay with a high signal-to-noise ratio. It has been applied to a test for the presence of ribosomal RNA of Campylobacter jejuni in biological samples.     

Application of pyronin Y(G) in cytochemistry of nucleic acids

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y(PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with double-stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 microM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.     

Non-radioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection-contrast and electron microscopy

A number of immunocytochemical detection systems for determining the chromosomal localization of specific nucleic acid sequences by non-radioactive in situ hybridization have been compared. The procedures were: 1. the peroxidase/diaminobenzidine (PO/DAB) combination, either or not gold/silver intensificated; 2. alkaline phosphatase marking using the nitro-blue tetrazolium plus bromochloro-indolyl phosphate substrate combination (AP/NBT + BCIP); and 3. immunogold with or without silver enhancement. The procedures were first tested and optimized in dot blot experiments and then applied to in situ hybridization. As hybridization probes, both a middle-repetitive and a unique sequence (modified with 2-acetylaminofluorene (AAF] were used. The advantages and disadvantages of the various methods for reflection contrast (RC) or transmission electron microscopic (TEM) visualization of hybrids are discussed.     

Reactions of the carcinogen N-acetoxy-4-acetamidostilbene with polynucleotides in vitro.

N-Acetoxy-4-acetamidostilbene (N-AcO-AAS) has been shown to react with mononucleosides to give numerous alkylation products [1]. In this work, homopolynucleotides, RNA and DNA were treated with N-[beta-14 C]-AcO-AAS, washed, degraded with S1 nuclease and acid phosphatase, and chromatographed on Sephadex LH-20. RNA prepared in vitro with 14C on cytosine, adenine or guanine was treated with non-radioactive N-AcO-AAS, then digested and chromatographed similarly. By this means, many of the adducts rising from nucleoside reactions were shown to result from treatment of nucleic acids with the same carcinogen, as well as a number of products which have not been matched to products of monomer alkylation. Labeled 1-(4-acetamidophenyl)-2-phenyl-1, 2-ethanediol was detected in the digest of RNA treated with radioactive N-AcO-AAS, suggesting that phosphate alkylation had taken place.     

Co-labeling using in situ PCR: a review.

In situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight DNase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1-10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive 3H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.     

A microassay for detection of DNA and RNA in small numbers of plant cells

Summary A microtechnique for the detection of DNA or RNA in small numbers of plant cells (1–50) has been developed using cauliflower mosaic virus (CaMV) infection of turnip as a model system. Both DNA and RNA extracted from 10 mesophyll protoplasts from CaMV-infected plants can be detected by hybridization using a radioactive probe made from cloned CaMV DNA (pCaMV10). No hybridization above background was detected in extracts of protoplasts from uninfected plants. At least 0.15 pg (11 000 molecules) of purified pCaMV10 DNA can be detected. This method is superior to existing macro techniques for nucleic acid detection as smaller amounts of tissue are required and the detection is approximately 100-fold more sensitive. re]19850326 rv]19850530 ac]19850611