mRNA-targeted fluorescent in situ hybridization (FISH) of Gram-negative bacteria without template amplification or tyramide signal amplification

Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multi-copy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact that there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate that the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.     

Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.

To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.     

Human papillomavirus detection by non isotopic in situ hybridization, in situ hybridization with signal amplification and in situ polymerase chain reaction

Classical in situ hybridization (ISH) with biotinylated probes makes it possible to detect and localize human papillomavirus (HPV) nucleic acid sequences in cytological and histological materials. This method is however of limited value in the detection of a few copies of the virus. Moreover the specificity of such a technique is not always convincing when ISH signals are small and/or of low intensity. Recently, much attention has been focused on the utility of the in vitro polymerase chain reaction (PCR) and especially on PCR-single strand conformation polymorphism (SSCP) to amplify small amounts of viral DNA with accurate hybrid specificity. But the latter method requires nucleic acid extraction and tissue destruction. Thus, correlation between the PCR results and histological findings is not possible. Hence, the aim of our current study was to apply to HeLa cells and cervical formalin-fixed and paraffin-embedded biopsies, a novel procedure of ISH signal amplification, the catalyzed signal amplification (CSA). Such a procedure is based on the deposition of streptavidin-horseradish peroxidase catalyzing the deposition of biotinylated tyramide molecules on the location of the probed target. The biotin accumulation is then detected with streptavidin peroxidase and diaminobenzidine. The results were compared with those obtained by direct and indirect in situ PCR. The catalysed signal amplification successfully increased the sensitivity and efficiency of ISH for the detection of rare sequences in HPV infected cells and histological materials. Such a method was found simpler and faster than in situ PCR and tissue morphology was better preserved.     

HPV DNA detection by in situ hybridization with catalyzed signal amplification on thin-layer cervical smears.

Thin layer-based technology in cervical cancer screening now allows both Papanicolaou staining and HPV testing on the same sample. Here, we show that in situ hybridization with catalyzed reporter deposition is a powerful HPV detection method when applied on thin-layer cervical smears, allowing distinction between two staining patterns suggestive of two different physical states of HPV DNA, where diffuse signals are suggestive of episomes and punctate signals are suggestive of viral DNA integration.     

Combined tyramide signal amplification and quantum dots for sensitive and photostable immunofluorescence detection.

Conventional immunofluorescence detection of biologically relevant proteins and antigens in tissue sections is often limited by relatively weak signals that fade rapidly on illumination. We have developed an immunohistochemical protocol that combines the sensitivity of tyramide signal amplification with the photostability of quantum dots to overcome these limitations. This simple method provides a sensitive and stable fluorescence immunohistochemical alternative to standard chromogen detection.     

Direct detection of 16S rRNA using oligonucleotide microarrays assisted by base stacking hybridization and tyramide signal amplification

A simple method has been developed and validated for direct, sensitive detection and specific identification of 16S rRNA. We first report our direct investigation of discrimination efficiency for sequence variations in RNA using oligonucleotide microarrays assisted by base stacking hybridization, and demonstrate that the sequence variations of double base substitution, single base substitution and single base deletion in RNA could be directly identified. With the help of tyramide signal amplification (TSA), the detection sensitivity of this method for four clinically important bacterial species was below 0.5, 5, 1 and 1 ng of total RNA, which are 100-1000 fold more sensitive than the published methods.     

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.     

Optimization of prostate cancer cell detection using multiplex tyramide signal amplification

Approximately 29 000 men die of prostate cancer (PCa) each year in the United States, and 90% to 100% of them are due to incurable bone metastasis. It is difficult to determine (1) when PCa disseminates in the natural history of the disease; (2) where cancer cell disseminates before becoming overt metastatic lesions; and (3) which tumors are aggressive and which are indolent. Tumor tissue and liquid (blood and bone marrow) biopsies provide important information to answer these questions, but significant limitations exist for immunostaining strategies that assess protein expression in these tissues. Classic immunohistochemistry (IHC) assays can typically assess expression of one or two proteins per tissue section. We have developed a novel immunofluorescence staining protocol to detect a panel of seven proteins on PCa tissue from primary tumor biopsies and metastatic lesion autopsy tissue, as well as cancer cells from liquid biopsies. We used a tyramide-based system to amplify the true signal and optimized the protocol to reduce background signal, thereby boosting the signal-to-noise ratio. Any protein-specific antibody in this protocol can be exchanged for a different validated antibody. This protocol therefore, represents a highly informative and flexible assay that can be used to provide important information about cancer tissue for the purpose of improving detection, diagnosis, and treatment.     

An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs.

In situ hybridization (ISH) using nonradioactive probes enables mRNAs to be detected with improved cell resolution but compromised sensitivity compared to ISH with radiolabeled probes. To detect rare mRNAs, we optimized several parameters for ISH using digoxygenin (DIG)-labeled probes, and adapted tyramide signal amplification (TSA) in combination with alkaline phosphatase (AP)-based visualization. This method, which we term TSA-AP, achieves the high sensitivity normally associated with radioactive probes but with the cell resolution of chromogenic ISH. Unlike published protocols, long RNA probes (up to 2.61 kb) readily permeated cryosections and yielded stronger hybridization signals than hydrolyzed probes of equivalent complexity. RNase digestion after hybridization was unnecessary and led to a substantial loss of signal intensity without significantly reducing nonspecific background. Probe concentration was also a key parameter for improving signal-to-noise ratio in ISH. Using these optimized methods on rat taste tissue, we detected mRNA for mGluR4, a receptor, and transducin, a G-protein, both of which are expressed at very low abundance and are believed to be involved in chemosensory transduction. Because the effect of the tested parameters was similar for ISH on sections of brain and tongue, we believe that these methodological improvements for detecting rare mRNAs may be broadly applicable to other tissues. (J Histochem Cytochem 47:431-445, 1999)     

Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification.

The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-monolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes.     

Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH)

Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.     

Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.     

Comparison of in-situ hybridization, direct and indirect in-situ PCR as well as tyramide signal amplification for the detection of HPV

 One hundred paraffin-embedded cervical biopsy specimens were tested for the presence of human papilloma virus (HPV) by in situ hybridization (ISH), and by direct and indirect in situ PCR (IS-PCR) in order to evaluate the efficiency of the different in situ methods in detecting HPV infection. ISH was performed using either commercial DNA probes or a cocktail of 5′-digoxigenin labeled oligoprimers. The same were used for ISH during indirect IS-PCR. To enhance the sensitivity of ISH several polymers, i.e., polyvinyl alcohol (PVA), polyethylene glycol, and polyvinylpyrrolidone were added to the alkaline phosphatase nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) reaction. Furthermore, tyramide signal amplification (TSA) was tried for signal amplification. Those samples treated with PVA during the NBT/BCIP reaction did not show any signal amplification whereas those treated with TSA exhibited a dramatic increase in sensitivity with usually acceptable signal to noise ratios. Our results show that, regarding sensitivity, ISH with subsequent signal amplification by TSA can be used as an almost equivalent alternative to the more cumbersome IS-PCR on routinely processed tissue specimens. When considering reproducibility, it is superior to IS-PCR. Accepted: 25 September 1998     

Chromosomal in situ hybridization with double-labeled DNA: signal amplification at the probe level.

A double-labeling approach was applied to nonisotopic in situ hybridization with individual cosmid and plasmid clones, using digoxigenin or biotin as label and a combination of two separate enzymatic labeling methods. Probe labeling was achieved by nick translation, followed by tailing of the probe by terminal deoxynucleotidyl transferase. The double-labeling method, in conjunction with an improved detection protocol, provides for a higher signal intensity than that obtainable with single-labeled probes.     

Group-specific quantification of methanotrophs in landfill gas-purged laboratory biofilters by tyramide signal amplification-fluorescence in situ hybridization

The aim of this study was to quantitatively analyse methanotrophs in two laboratory landfill biofilters at different biofilter depths and at temperatures which mimicked the boreal climatic conditions. Both biofilters were dominated by type I methanotrophs. The biofilter depth profiles showed that type I methanotrophs occurred in the upper layer, where relatively high O(2) and low CH(4) concentrations were present, whereas type II methanotrophs were mostly distributed in the zone with high CH(4) and low O(2) concentrations. The number of type I methanotrophic cells declined when the temperature was raised from 15 degrees C to 23 degrees C, but increased when lowered to 5 degrees C. A slight decrease in type II methanotrophs was also observed when the temperature was raised from 15 degrees C to 23 degrees C, whereas cell numbers remained constant when lowered to 5 degrees C. The results indicated that low temperature conditions favored both type I and type II methanotrophs in the biofilters.     

Simultaneous detection of six human diarrheal pathogens by using DNA microarray combined with tyramide signal amplification

Multiplex PCR and DNA microarray were combined with tyramide signal amplification (TSA) to develop a reliable method suitable for simultaneous detection of six species of human diarrheal pathogens (Yersinia enterocolitica, Shigella spp, Salmonella typhi, Brucella spp, Vibrio cholera and Escherichia coli O157:H7). Meanwhile, our method could distinguish V. cholera serotype O1 from O139, and O157:H7 from O157: non-H7. This assay conferred a specificity of 100% for target pathogens. The limit of detection was 103 degrees CFU/mL approximately. The results of 98.6% (357/362) clinical specimens and 100% (5/5) mocked double-blind samples were the same to that from conventional assay. Consequently this assay is sensitive and a specific tool suitable for diagnostic detection and surveillance of multiple human pathogens.