Chemiluminescent assay for the detection of viral and plasmid DNA using digoxigenin-labeled probes.

A dot-blot hybridization immunoenzymatic assay with a chemiluminescent endpoint was developed for the rapid and sensitive detection of viral and plasmid DNAs. Digoxigenin-labeled probes were used to detect cytomegalovirus, parvovirus B19, and plasmid pBR328 DNAs. Hybridized probes were immunoenzymatically visualized by anti-digoxigenin Fab fragments labeled with alkaline phosphatase, and adamantyl 1,2-dioxetane phenyl phosphate was used as chemiluminescent substrate. Results were recorded by instant photographic films. The chemiluminescent hybridization assay was performed in about 8 hr and was able to detect as little as 50-10 fg of homologous target DNA.     

Gene rearrangements detected by nonradioactive digoxigenin-labeled DNA probes.

The configurations of immunoglobulin, T-cell receptor beta chain and bcl-2 genes were analyzed in lympho-proliferative disorders using nonradioactive digoxigenin-labeled probes. The studies demonstrated the reliability of digoxigenin-labeled probes for the detection of single-copy genes after Southern blotting of genomic DNA: 1 microgram and sometimes even 0.2 microgram of restriction endonuclease-digested DNA could be detected either by JH, C beta or bcl-2 probes. The intensity of the signal was consistently satisfactory, and there was no background problem. Control experiments showed neither false-positive nor false-negative results caused by the use of the nonradioactive detection system.     

Polysaccharide synthesis in tissue sections

Summary Biochemical methods for demonstration of enzyme activity are test tube models outside the organization of cell. Their application to the complicated organization of the cell present problems to histochemistry. The morphological and chemical preservation of tissue which is desirable in histochemistry leads to a multiplicity of reactions when test tube methods are applied. For example, the histochemical phosphorylase and glycosyltransferase reactions rest on the assumption that one can distinguish between preexisting glycogen and newly formed polysaccharides. We used frozen dried canine myocardium and liver for examination of the authenticity of histochemical phosphorylase and glycosyltransferase (branching enzyme, UDPG-glycogen transglycosylase) reactions as described in histochemical reference books. We were unable to distinguish between preexisting glycogen and supposedly newly formed polysaccharides with methods presently used for this purpose (Iodine stain, differential digestion with amylases, acid hydrolysis). Tissue without PAS stainable glycogen remained so after substrate incubation. When preexisting glycogen was present, the amounts of stainable polysaccharides after incubation were invariably less. Therefore, we could not prove beyond doubt that any polysaccharide synthesis due to enzyme reaction had oceured. The prescribed controls, perhaps adequate for biochemical test tube reactions, have to be redefined for meaningful histochemical procedures.This work was supported by grant number HE-07605-07 from the National Heart Institute, National Institutes of Health, Bethesda, Md., U.S.A.     

Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification

The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.     

Fluorescent in situ hybridization en suspension (FISHES) using digoxigenin-labeled probes and flow cytometry.

Fluorescent in situ hybridization has become a major technique for visualizing genetic material in fixed cells. Currently, many systems utilize the hybridization of labeled molecular probes to cells that are attached to slides. We have developed a technique that allows for in situ hybridization to be performed using cells in suspension. By using digoxigenin-labeled DNA probes and a fluoresceinated antibody directed against the digoxigenin, we can measure the resulting signal on a flow cytometer and the cells can be attached to microscope slides for visual analysis.     

Detection of apoptosis in tissue sections

During the last few years, detection of apoptosis has evolved from a predominantly morphological basis to the use of ever more specific techniques. The methods widely used to visualize DNA fragmentation in tissue sections are now supplemented by a variety of specific antisera against components of the cell death pathways. Essential requirements for apoptosis detection techniques include high sensitivity for apoptotic cells, the ability to differentiate between apoptotic and necrotic cell death and other forms of DNA damage, and the distinction between different stages of the cell death process. In this overview, we will focus on recent technical advances in apoptosis detection covering improvements of in situ DNA fragmentation techniques, as well as pointing out some of the new tools available for the detection of apoptotic cells in tissue.     

Detection of human adenoviruses with real-time PCR assay using TaqMan fluorescent probes

Infections with human adenoviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from A549 cell line infected with five different HAdV strains was used for development of a qualitative real-time PCR assay for detection of all human adenoviruses using primers targeting a conserved region of the hexon gene and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HAdV7 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for adenovirus detection with the same DNA dilutions was made. The sensitivity of novel method; was about thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with adenoviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of adenoviral DNA in clinical specimens, especially from neuroinfections or immunocompromised hosts.     

Real-time PCR genotyping using displacing probes

Simple and reliable genotyping technology is a key to success for high-throughput genetic screening in the post-genome era. Here we have developed a new real-time PCR genotyping approach that uses displacement hybridization-based probes: displacing probes. The specificity of displacing probes could be simply assessed through denaturation analysis before genotyping was implemented, and the probes designed with maximal specificity also showed the greatest detection sensitivity. The ease in design, the simple single-dye labeling chemistry and the capability to adopt degenerated negative strands for point mutation genotyping make the displacing probes both cost effective and easy to use. The feasibility of this method was first tested by detecting the C282Y mutation in the human hemochromatosis gene. The robustness of this approach was then validated by simultaneous genotyping of five different types of mutation in the human β-globin gene. Sixty-two human genomic DNA samples with nine known genotypes were accurately detected, 32 random clinical samples were successfully screened and 114 double-blind DNA samples were all correctly genotyped. The combined merits of reliability, flexibility and simplicity should make this method suitable for routine clinical testing and large-scale genetic screening.     

A new method to perform nonradioactive tissue printing using digoxigenin-labeled riboprobes

We describe a modified protocol to perform a tissue print northern using digoxigenin-labeled riboprobes. The result of the hybridization is directly visualized on the membrane by an alkaline phosphatase detection system.     

Detection of altered retinoic acid receptor expression in tissue sections using in situ hybridization

Nuclear retinoid receptors mediate retinoid effects in controlling cell growth, differentiation, apoptosis, and carcinogenesis. Altered expression or activity of these receptors could abolish the retinoid signal transduction pathway and be associated with human carcinogenesis. In situ hybridization is a powerful tool for analyzing gene expression in formalin-fixed, paraffin-embedded tissue sections, especially for newly cloned genes or when no antibodies are available. Detection of altered retinoid receptor expression using in situ hybridization in premalignant and malignant tissues has provided important information about the roles of these receptors in cancer development and the response of these tissues to retinoid treatment. Among these receptors, altered expression of retinoic acid receptor-beta (RAR-beta) has been mostly detected in human cancers, including those of the head and neck, lung, esophagus, mammary gland, pancreas, and cervix. RAR-beta is thus currently used as a surrogate endpoint biomarker in different clinical prevention trials of various cancers.     

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.     

Use of non-radioactive digoxigenin-labeled DNA probes for RFLP analysis in rice

A simple procedure for the detection of rice RFLPs with non-radioactive probes is described. Rice single-copy DNA was labeled with non-radioactive digoxigenin-d UTP. When digested total DNA was hybridized with the non-radioactive labeled DNA probes, RFLPs for rice single-copy DNA could be successfully detected.     

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).     

Cancer cell detection in tissue sections using AFM

Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Young's modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.     

Detection of gastrin-specific mRNA using oligodeoxynucleotide probes of defined sequence.

Three oligodeoxynucleotides have been chemically synthesized and used as hybridization probes to detect gastrin-specific mRNA within an heterogeneous population of RNA molecules. Using these oligonucleotides whose nucleotide sequences were deduced from the amino acid sequence of the hormone, 0.2 fmol of gastrin mRNA can be detected/microgram of poly(A)-RNA from hog antrums. The 32P-labeled oligonucleotides hybridize specifically to RNA covalently coupled to DBM-paper (Alwine, J.C., Kemp, D.J., and Stark, G.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354). Labeled oligonucleotides also hybridize specifically to RNA separated by gel electrophoresis in the presence of CH3HgOH and covalently coupled to DBM-paper. Using this procedure, we have found that the mRNA coding for gastrin contains about 620 nucleotides, which is in agreement with results obtained using gastrin cDNA to determine mRNA size.     

In situ hybridization: use of 35S-labeled probes on paraffin tissue sections

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.