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.     

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.     

Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.     

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a ³⁵S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascinspecific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.     

A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2��-O-Methyl RNA + LNA

The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low–copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2′-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA–based probes and the optimized ISH assay enable simple and fast detection of low–copy number miRNA targets, such as miR-130a in mouse brain.     

Fluorescence and chromogenic in situ hybridization to detect genetic aberrations in formalin-fixed paraffin embedded material, including tissue microarrays

Screening for specific genetic aberrations by fluorescence and chromogenic in situ hybridization (fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH)) can reveal associations with tumor types or subtypes, cellular morphology and clinical behavior. FISH and CISH methodologies are based on the specific annealing (hybridization) of labeled genomic sequences (probes) to complementary nucleic acids within fixed cells to allow their detection, quantification and spatial localization. Formalin-fixed paraffin embedded (FFPE) material is the most widely available source of tumor samples. Increasingly, tissue microarrays (TMAs) consisting of multiple cores of FFPE material are being used to enable simultaneous analyses of many archival samples. Here we describe robust protocols for the FISH and CISH analyses of genetic aberrations in FFPE tissue, including TMAs. Protocols include probe preparation, hybridization and detection. Steps are described to reduce background fluorescence and strip probes for repeat FISH analyses to maximize the use of tissue resources. The basic protocol takes 2-3 d to complete.     

Visualization of gene expression in whole mouse retina by in situ hybridization

The mouse retinal vasculature provides a powerful model system for studying development and pathologies of the vasculature. Because it forms as a two-dimensional flat plexus, it is easily imaged in its entirety in whole-mount retinal preparations. In order to study molecular signaling mechanisms, it is useful to visualize the expression of specific genes in the entire vascular plexus and retina. However, in situ hybridization on whole-mount retinal preparations is problematic because isolated retinas have a tendency to curl up during hybridization and are difficult to stain. Here we provide a detailed protocol that overcomes these difficulties and visualizes the mRNA distribution of one or two genes in the context of the counterstained retinal vasculature. The protocol takes 3-4 d for single-probe stains, with an additional 2 d for immunohistochemistry co-labeling. In situ hybridization with two probes adds a further 3 d.     

A protocol for PCR in situ hybridization of hyphomycetes.

A protocol for application of Polymerase Chain Reaction (PCR) in situ hybridization for the detection of hyphomycetes is presented. The experiments are exemplary carried out with strains of the genera Penicillium and Cladosporium. The small ribosomal subunit is amplified in situ by PCR using fungal specific primers. The amplicon is used as target region for a fluorescein-marked probe. The permeability of the fungal cell wall for the primers and the probe can be successfully achieved by enzymatic treatment with beta-glucanase. The protocol can be used as a basis for further development of in situ hybridization with taxon specific probes.     

BAC 'landing' on chromosomes of Brachypodium distachyon for comparative genome alignment

Fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) with large genomic DNA inserts as probes (BAC 'landing') is a powerful means by which eukaryotic genomes can be physically mapped and compared. Here we report a BAC landing protocol that has been developed specifically for the weedy grass species Brachypodium distachyon, which has been adopted recently by the scientific community as an alternative model for the temperate cereals and grasses. The protocol describes the preparation of somatic and meiotic chromosome substrates for FISH, the labeling of BACs, a chromosome mapping strategy, empirical conditions for optimal in situ hybridization and stringency washing, the detection of probes and the capturing and processing of images. The expected outcome of the protocol is the specific assignment of BACs containing single-copy inserts to one of the five linkage groups of the genome of this species. Once somatic or meiotic material is available, the entire protocol can be completed in about 3 d. The protocol has been customized empirically for B. distachyon and its near relatives, but it can be adapted with minor modifications to diverse plant species.     

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (> 98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences.     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

Histological aspects of in situ hybridization. Detection of poly(A) nucleotide sequences in mouse liver sections as a model system

This study examined the detection of cellular poly(A) sequences in mouse liver sections by in situ hybridization using a 3H-labelled poly(dT) probe. Parameters examined included possible losses of target poly(A) sequences from sectioned cells, access of probe to target sequences, section thickness, hybridization conditions, autoradiographic efficiency, specific activity of probes and specificity of reaction. An improved protocol was devised that resulted in good preservation of histological detail in sectioned tissue blocks, and a calculated hybridization efficiency of 50%-100%. With the use of probes of defined sequence, the protocol should allow detection of unique mRNA sequences within single cells with an estimated sensitivity of 6-12 unique mRNA molecules per sectioned cell.