Double and triple in situ hybridization for coexpression studies: combined fluorescent and chromogenic detection of neuropeptide Y (NPY) and serotonin receptor subtype mRNAs expressed at different abundance levels

Multiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance in the regions analyzed. However, expression of 5-HT1A mRNA, which is expressed at comparatively low abundance in many telencephalic areas, could not be unequivocally identified in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in fluorescent receptor mRNA detections. Parallel chromogenic in situ hybridization provided clear labeling for 5-HT1A mRNA and additionally offered the possibility to monitor the chromogen deposition at regular time intervals to determine the optimal signal-to-noise ratio. We first developed a double labeling protocol combining fluorescence and chromogenic in situ hybridization and subsequently expanded this variation to combine double fluorescence and chromogenic in situ hybridization for triple labelings. With this method, we documented expression of 5-HT2C and/or 5-HT1A in subpopulations of telencephalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and fluorescence microscopy, combines advantages of fluorescence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to previously published multiple labeling methods for coexpression analyses in which one mRNA species requires highly sensitive detection.     

Prolonged hybridization with a cRNA probe improves the signal to noise ratio for in-tube in situ hybridization for quantification of mRNA after fluorescence-activated cell sorting

We developed an in-tube in situ hybridization method for mRNA quantification after fluorescence-activated cell sorting (FACS-mQ). A specific RNA in a particular cell type is stained with a cRNA probe and a fluorescent dye, which allows the stained cells to be selected by FACS without excessive RNA degradation. Our previous protocol required 4 h for hybridization with a cRNA probe, which might not produce enough fluorescence signal for sorting genes with low expressions. We determined the effect of prolonged hybridization for in-tube in situ hybridization on quantitative measurement of intracellular RNAs. During the hybridization step, the quantity of ACTB mRNA decreased gradually until 4 h, but remained constant from 4 to 16 h below 63.6° C. For flow cytometry, cells hybridization with cRNA probes for TG mRNA at 60° C for 16 h showed both increased signal and decreased background fluorescence compared to those hybridized for 4 h. These results indicate that when performing in-tube in situ hybridization, hybridization temperature can be raised to 63.6° C and the hybridization step can be extended up to 16 h without excessive intracellular RNA degradation.     

mRNA localization in polarized intestinal epithelial cells

An important feature of enterocyte maturation is the asymmetrical distribution of cellular functions including protein localization. mRNA sorting is one mechanism for establishment and maintenance of this process in other systems, and we have previously demonstrated differential localization of mRNAs in human enterocytes. To study regulation of mRNA sorting, we established a model in polarized Caco-2 cells. Proxy cDNA constructs containing beta-galactosidase (beta-gal)/green fluorescence protein (GFP) and the 3'-untranslated region (3'-UTR) of either human sucrase-isomaltase or villin were transfected transiently or stably. A control construct contained poly-A sequence in place of 3'-UTR. Expression of GFP was observed by confocal microscopy; intracellular location of the construct mRNA was imaged by in situ hybridization. The sucrase-isomaltase mRNA proxy localized to an apical position in Caco-2 cells as in native enterocytes; the villin mRNA proxy did not show significant localization. The control construct was not localized and was found diffusely throughout the cell. Proxy GFP proteins tended to localize with their mRNA proxies, but with less precision. This study establishes a valuable model for the investigation of mRNA localization in intestinal epithelial cells. Mechanisms controlling asymmetrical distribution of intestinal mRNAs can be now be elucidated.     

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.     

In situ hybridization in suspension and flow cytometry as a tool for the study of gene expression.

We describe a method to detect mRNA expression using in situ hybridization in suspension and flow cytometry. Our model was glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in the leukemic cell line K562. A GAPDH cDNA probe was labeled with digoxigenin-11-dUTP and detected using an FITC-labeled anti-digoxigenin antiserum. We obtained good resolution in specific signals against background (GAPDH signal/control plasmid signal ratio +/- SE 3.5 +/- 0.9). The technique was optimized taking into account several hybridization variables, like fixation, hybridization time, effect of blocking agents, and stringency wash variations. This method also allowed us to quantitate the GAPDH RNA copy number/cell using a fluorescence standard; we obtained a figure of about 1200 copies/cell, which is in good agreement with the dot blot hybridization assay result. Flow cytometric analysis of in situ hybridization represents an original method to study gene expression. This technique has the potential to develop into a multiparametric tool for cell biology studies, examining specific mRNA production together with DNA content or membrane molecules expression, and offering the possibility to purify by sorting a cell population expressing a specific mRNA.     

Improved procedure for fluorescence in situ hybridization on tissue microarrays

BACKGROUND: The recently developed tissue microarray (TMA) technology allows the arrangement of up to a thousand tissue specimens on a single microscope slide. This technology enables researchers to perform gene copy number studies on very large series of archival formalin-fixed tissues using fluorescence in situ hybridization (FISH). However, the hybridization properties of individual archival specimens can vary considerably. Therefore a highly optimized protocol is needed to fulfill the task of producing evaluable hybridization signals simultaneously in hundreds of specimens in a TMA. METHODS: The performance of two different FISH protocols, the standard protocol for paraffin embedded tissues and our new optimized protocol, was tested on TMAs using probes for the HER-2 and ZNF217 genes as well as the chromosome 17 centromere. RESULTS: The new protocol resulted in greatly increased signal intensity and an almost 30% increase in the number of tissue samples with evaluable hybridization signals. CONCLUSIONS: Our improved protocol for FISH on TMAs provides standardized hybridization conditions leading to high-quality hybridization signals in the majority of specimens. The increases in the signal intensity and the number of evaluable samples are extremely important for the successful analyses of TMAs by FISH and will allow the utilization of the TMA technology in its full potential.     

Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons

The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies. However, the mRNA detection specificity and sensitivity are critically dependent on the selection of target sequences and their accessibility. We carried out an extensive study of the target accessibility of BMP-4 mRNA using 10 different designs of molecular beacons (MBs), and identified the optimal beacon design. Specifically, for MB design 1 and 8 (MB1 and MB8), the fluorescent intensities from BMP-4 mRNA correlated well with the GFP signal after upregulating BMP-4 and co-expressing GFP using adenovirus, and the knockdown of BMP-4 mRNA using siRNA significantly reduced the beacon signals, demonstrating detection specificity. The beacon specificity was further confirmed using blocking RNA and in situ hybridization. We found that fluorescence signal from MBs depends critically on target sequences; the target sequences corresponding to siRNA sites may not be good sites for beacon-based mRNA detection, and vice versa. Possible beacon design rules are identified and approaches for enhancing target accessibility are discussed. This has significant implications to MB design for live cell mRNA detection.     

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.     

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.     

Direct and sensitive fluorescence in situ hybridization of 45S rDNA on tomato chromosomes.

We describe a direct and sensitive fluorescence in situ hybridization protocol for plant chromosomes. We labelled 45S rDNA with fluorescein-12-dUTP and hybridized to somatic chromosomes of four tomato genotypes. This technique does not require posthybridization immunocytochemical amplifications. The improved signal sensitivity with this technique allowed identification of new rDNA loci on three pairs of chromosomes, in addition to the previously known locus on chromosome 2. We discuss favorable features of direct fluorescence in situ hybridization for chromosomes fixed on a slide and chromosomes or cells in suspension.     

Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.     

Validation and development of quantitative flow cytometry-based fluorescence in situ hybridization for intercenter comparison of telomere length measurement

BACKGROUND: Telomeres are highly conserved repeats at the ends of chromosomes that maintain chromosome stability and reflect the replicative potential of cells. Telomere length can be determined by Southern blot hybridization or quantitative fluorescence in situ hybridization (Q-FISH). Recently, two flow cytometry-based (Flow) FISH protocols have been published. METHODS: We compared the telomere length measured by Southern blotting and Flow FISH using standard beads to calibrate and quantify the fluorescence intensity. RESULTS: The telomeric fluorescence of cord blood and peripheral blood mononuclear cells was similar to that reported by other studies. There was a linear relationship between the telomeric fluorescence determined by Flow FISH and the telomere fragment size determined by Southern blotting (r = 0.89; P < 0.001). CONCLUSION: It is important to set up a center-specific curve and select appropriate cell lines for reference. This Q-Flow FISH protocol will facilitate the measurement of telomere length and allow more meaningful comparison of data (in standard fluorescence units or fragment size) between institutes.     

Histological aspects of in situ hybridization

Summary 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, autoradioigraphic 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.