Photo-cross-linkable oligonucleotide probes for in situ hybridization assays

In situ hybridization techniques have been an important research tool since first introduced 30 years ago, and more recently clinical applications have been expanding greatly. Still, further improvements in the assay sensitivity and protocols that are amenable to routine clinical use are desired. We use a novel photo-cross-linking technology to irreversibly bind short oligonucleotide probes to the target sequence following a hybridization period. The cross-linking agent is incorporated into the backbone of the probe and is activated to react with pyrimidines in the opposite strand by near-UV (300-370 nm) irradiation. By locking the probe to the target, very stringent wash conditions can be used that would otherwise completely remove probes that are hybridized but not cross-linked to the target. Consequently, the probe-specific signal is maximized, while the background signal is minimized to the greatest extent possible with the stringency of the wash. The use of short, photo-cross-linkable probes presents a new strategy for maximizing the sensitivity of probe hybridization or signal amplification-based in situ techniques.     

Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry.

Because of their tiny size (0.2 to 2 microns), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features.     

Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes

In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs ( approximately 20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have described a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here, we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression.     

Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ

Summary A fast, reproducible and non-hazardous technique for non-isotopic DNA fingerprinting is presented. The method is based on digoxigenated oligonucleotides, which are specific for simple repetitive DNA sequences. The use of digoxigenin/ anti-digoxigenin detection avoids many drawbacks inherent in e.g. the biotin/streptavidin system which often causes a poor signal-to-background ratio. Synthesis and purification of digoxigenated oligonucleotides and their use in filter hybridization are described in detail. Hybridization patterns obtained with four different radioactively labeled oligonucleotides have been compared with those of the respective digoxigenated probes. When slightly less stringent hybridization conditions are applied for digoxigenated oligonucleotides than for those labeled with ³²P, the signal intensities are satisfying but additional minor bands occur as a result of the reduced strigency. With one explainable exception, these bands increase the information content of the fingerprint. In addition, hybridization of the digoxigenated (CAC)₅ probe has been performed in situ with human metaphase chromosomes. The hybridization patterns in many mitoses resemble R-bands.     

Colorimetric in situ hybridization (CISH) with digoxigenin-labeled oligonucleotide probes in autofluorescent hyphomycetes.

We used digoxigenin-labeled probes for in situ hybridization of hyphomycetes to replace the commonly used fluorescent proof of probe binding by a colorimetric reaction. The resulting blue-purple, intracellular precipitate could be easily detected by light microscopy, and thus presented a promising method to overcome autofluorescence of fungal material and substratum. Optimal cell fixation and permeabilization procedures, as well as hybridization conditions were developed on the example of two different hyphomycetes: Phialophora sp. and Cartapip, a colorless mutant of Ophiostoma piliferum (Agra Sol).     

Immunocytochemistry of 5-bromo-2'-deoxyuridine labelled oligonucleotide probes. A novel technique for in situ hybridization

A synthetic oligonucleotide probe, complementary to oxytocin m-RNA was labelled enzymatically with 5-bromo-2'-deoxyuridine (5-BrdU) and with [gamma-32P]-ATP. The labelled probes were used for in situ hybridization of histological sections of the mouse hypothalamus. A monoclonal antibody to 5-BrdU and the streptavidine-peroxidase technique were used in order to visualize hybridization with the 5-BrdU labelled probe. In situ hybridization with [32P] labelling was detected autoradiographically. With both methods hybridized neurons were visible in the magnocellular hypothalamic nuclei. While immunostaining and radio-labelling provided similar localization of oxytocin m-RNA, only the immunocytochemical technique showed clear cellular resolution of the reaction product. In situ hybridization with 5-BrdU labelled probes followed by 5-brdU immunocytochemistry seems to be a powerful alternative to common autoradiographic techniques.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

Summary We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electronmicroscopical level.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding reserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electron-microscopical level.     

Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phosphatase-labeled oligonucleotide probe.

An oligonucleotide DNA probe (VVAP) was constructed from a portion of the Vibrio vulnificus cytolysin gene (hylA) sequence and labeled with alkaline phosphatase covalently linked to the DNA. Control and environmental isolates probed with VVAP showed an exact correlation with results obtained with a plasmid DNA probe (derived from pCVD702) previously described as having 100% specificity and sensitivity for this organism. Identification of V. vulnificus strains was confirmed independently by analysis of the cellular fatty acid composition and by API 20E. Naturally occurring V. vulnificus bacteria were detected without enrichment or selective media by VVAP in unseeded oyster homogenates and seawater collected from a single site in Chesapeake Bay during June at concentrations of 6 x 10(2) and 2 x 10(1) bacteria per ml, respectively. V. vulnificus bacteria were also enumerated by VVAP in oysters seeded with known concentrations of bacteria and plated on nonselective medium. The VVAP method provides a rapid, accurate means of identifying and enumerating V. vulnificus in seawater and oysters without the use of selective media or additional biochemical tests.     

The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.     

Non-radioactive in situ hybridization for mRNA with emphasis on the use of oligodeoxynucleotide probes

 Synthetic oligodeoxynucleotides represent attractive tools for mRNA in situ hybridization. In the present review, guidelines for the preparation, labeling and use of such probes is discussed. Emphasis is placed on sensitivity and specificity issues. Accepted: 20 June 1997     

Cooperativity of paired oligonucleotide probes for microarray hybridization assays

Synthetic DNA probes attached to microarrays usually range in length from 25 to 70 nucleotides. There is a compromise between short probes with lower sensitivity, which can be accurately synthesized in higher yields, and long probes with greater sensitivity but lower synthesis yields. Described here are microarrays printed with spots containing a mixture of two short probes, each designed to hybridize at noncontiguous sites in the same targeted sequence. We have shown that, for a printed microarray, mixed probe spots containing a pair of 30mers show significantly greater hybridization than spots containing a single 30mer and can approach the amount of hybridization to spots containing a 60mer or a 70mer. These spots with mixed oligonucleotide probes display cooperative hybridization signals greater than those that can be achieved by either probe alone. Both the higher synthesis yields of short probes and the greater sensitivity of long oligonucleotides can be utilized. This strategy provides new design options for microarray hybridization assays to detect RNA abundance, RNA splice variants, or sequence polymorphisms.     

Preparation of nonradioactive probes for in situ hybridization

In situ hybridization (ISH) enables the precise localization of RNA targets and provides an avenue to study the temporal and spatial patterns of expression of specific genes. ISH has evolved from being an esoteric technique to one that is routinely used by researchers in many areas of research. A major driving force has been the development of numerous nonisotopic labeling and signal detection methods. Historically, radioactive probes and autoradiography provided sensitivity that was unattainable with nonisotopic probes. But the long exposure times required for signal detection and the perceived dangers associated with radioactivity limit its use. Advances in nonisotopic detection systems have overcome many of the limitations associated with using radiolabeled probes. One of the most significant contributions from nonisotopic methods is the ability to discriminate between multiple nucleic acid sequences simultaneously.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

The use ofin situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain

1. The application of in situ hybridization histochemistry to the study of neuropeptide gene expression in human brain postmortem tissues is reviewed. We focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia. 32P-labeled oligonucleotides were used as hybridization probes. 2. Autoradiography combined with computerized image analysis was used to visualize and quantify the hybridization signal. 3. Several criteria were considered in order to ascertain the specificity of the signal, including Northern analysis, use of heterologous probes, competition assays, and thermal stability of the hybrids. 4. In control human striatum high levels of hybridization signal were observed for somatostatin, neuropeptide Y, and preproenkephalin A mRNAs. In contrast, no detectable signal was observed with the cholecystokinin, arginine-vasopressin, and oxytocin probes in this area. In the hypothalamus high levels of oxytocin and arginine-vasopressin mRNAs were visualized in several nuclei. Preproenkephalin A and somatostatin mRNAs were also observed in this region, while cholecystokinin mRNA was not detected. 5. No significant correlations were found between the density of the hybridization signal and parameters such as postmortem delay, age, and gender in the population studied. 6. Finally, alterations of mRNA levels for some of these peptides were found in Parkinson's disease and Huntington's chorea striatal tissues. 7. These results show that in situ hybridization histochemistry can be used to examine at the microscopic level neuropeptide gene expression in postmortem materials.