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.     

昆虫小器官RNA荧光原位杂交技术

【目的】在昆虫基因表达和功能研究中,RNA原位杂交技术越来越受到青睐。该技术不仅能定性定量反应基因表达的时空特异性,而且能在细胞水平上检测基因表达的调控模式。为了将该技术更好地在昆虫小器官研究中运用,我们以果蝇幼虫翅芽为例优化了改技术。【方法】解剖果蝇3龄幼虫翅芽进行原位杂交实验。【结果】我们发现影响原位杂交结果的因素十分复杂,包括取材时期,探针的合成,预杂交/杂交的时间和温度,清洗时间,适当的对照等。通过RNA荧光原位杂交实验,我们揭示了调控细胞记忆的trithorax基因在3龄翅芽广泛表达,并且受到转录因子Optomotor-blind的负调控。【结论】这一技术方法为研究昆虫小器官的基因表达和调控提供了便捷手段。     

Highly fluorochrome labeled gene probes for quantitative tracing of RNA in individual cells by in situ hybridization.

A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochrome-labeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeled gene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation.     

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.     

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.     

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.     

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.     

Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture

We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.     

The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function

A new microarray method, the isotope array approach, for identifying microorganisms which consume a (14)C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [(14)C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of (14)C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO(2) fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [(14)C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO(2) fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by (14)C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of (14)C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

Application and validation of DNA microarrays for the 16S rRNA-based analysis of marine bacterioplankton

An oligonucleotide probe-based DNA microarray was evaluated for its ability to detect 16S rRNA targets in marine bacterioplankton samples without prior amplification by polymerase chain reaction (PCR). The results obtained were compared with those of quantitative fluorescence in situ hybridization (FISH). For extraction and direct labelling of total RNA, a fast and efficient protocol based on commercially available kits was established. A set of redundant and hierarchically structured probes was applied, and specificity of hybridization was assessed by additional control oligonucleotides comprising single central mismatches. The protocol was initially tested by microarray analysis of bacterial pure cultures. Complete discrimination of all control oligonucleotides was achieved, indicating a high degree of hybridization specificity. In a co-culture, abundant members were detected by microarray analysis, but signal ratios of positive probes did not correlate well with quantitative data from FISH experiments. A marine picoplankton sample from the German Bight was analysed. Bacterial populations with relative abundances of at least 5% were detected by hybridizing 0.1 microg of total RNA extracted from a sample of 375 ml equivalent to 4.1 x 10(8) cells. Our results demonstrate that major populations of marine bacterioplankton can be identified by microarray analysis in a fast and reliable way, even in relatively low volumes of sea water.     

mRNA Quantification After Fluorescence Activated Cell Sorting Using Locked Nucleic Acid Probes

Recently, we have established an in-tube in situ hybridization method named mRNA quantification after fluorescence activated cell sorting (FACS-mQ), in which a specific RNA in a particular cell type is stained with a florescent dye, allowing the stained cells to be selected by FACS without suffering excessive RNA degradation. Using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we used locked nucleic acid (LNA) oligonucleotides, which are known to enhance both the sensitivity and specificity of RNA detection, as hybridization probes in FACS-mQ. When we used a LNA probe targeting the human 28S sequence, we were able to efficiently separate human cells from rat cells. Using LNA probes, the hybridization step was shortened to 1 h. After the hybridization step, 84.6% RNA was preserved; thus, we were able to successfully measure gene expression levels in each type of cell after FACS. Providing the LNA probe efficiently hybridizes with the target sequence, FACS-mQ with an LNA probe is a powerful tool for separating particular cells and determining their biological characteristics by analyzing their gene expression profile.