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PubMed2005

Graphical representation of ribosomal RNA probe accessibility data using ARB software package.

BACKGROUND: Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ hybridization (FISH), besides single cell identification, allows the localization and functional studies of the microbial community composition. Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments. RESULTS: The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns. The different types of sequence associated information (SAI) can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool. CONCLUSION: Using this tool, in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of in situ hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page http://www.arb-home.de

Algorithms↗
PubMed1989

Differentiation-linked human papillomavirus types 6 and 11 transcription in genital condylomata revealed by in situ hybridization with message-specific RNA probes.

Human papillomaviruses (HPVs) infect specific human epithelial tissues. Because viral propagation in cultured cells has not been achieved, studies of HPV genetic activities have been difficult and rely largely on analyses of patient specimens by conventional biochemical methods. HPV type 6 and type 11 infections often result in genital warts (condylomata acuminata). Structural mapping of RNAs from such warts reveals that they use alternative promoters, splice sites, and polyadenylation sites to produce complex families of overlapping mRNAs that span multiple open reading frames. Based on the mRNA structures, we have developed message-specific subgenomic clones of HPV-6 and HPV-11 in pGEM vectors. Tritium-labeled, single-stranded RNA probes were synthesized in vitro and applied to serial thin sections of patient specimens for in situ hybridization. Our results reveal the qualitative and quantitative transcription patterns of different viral messages in relationship to one another, to viral DNA replication, and to cellular differentiation. The viral "E region" is transcribed before the onset of vegetative DNA replication and continues to be expressed in increasing amounts in the maturing epithelium. Even in mature epithelia, E region messengers are far more abundant than "L region" mRNAs. The L region messages encoding capsid proteins are truly late in that they appear concomitant with or after the onset of vegetative viral DNA replication and are only present in the superficial strata of the epithelium, which contain the oldest and most differentiated keratinocytes. Abundant intron material derived from processing E region transcripts accumulates in the nuclei. Strictly nuclear signals from the L region transcripts in the midepithelium suggest that regulation of their expression is at the level of transcription elongation.

Condylomata Acuminata↗
PubMed1993

Single-stranded RNA probes generated from PCR-derived DNA templates.

The following report outlines the use of the polymerase chain reaction (PCR) in combination with in vitro transcription to generate single-stranded radiolabelled RNA probes useful for nuclease protection and in situ hybridization experiments. Specific DNA fragments with bacteriophage promoter (T3 and/or T7) sequences at the 5' or 3' end are generated by repeated rounds of amplification. Following purification, these PCR-generated DNA products are used as templates for in vitro transcription with the correct DNA-dependent RNA polymerase. The resultant radiolabelled, single-stranded RNA (ssRNA) can be used for in situ hybridization, Southern or Northern blot analysis, and ribonuclease protection experiments. Sub-cloning or hydrolysis of large fragments is not required. Probes can be made from virtually any sequence using a variety of template sources.

Animals↗
PubMed1992

Detection of Listeria monocytogenes by non-radioactive RNA probe and polymerase chain reaction.

A HindIII fragment, 651-base-pair, from the listeriolysin 0 gene of Listeria monocytogenes was cloned into pGEM4Z. Radioactive 32P labeled and non-radioactive digoxigenin labeled RNA probes were made by in vitro transcriptions. These probes hybridized only to L. monocytogenes strains. Polymerase chain reaction was performed by using a 22-mer and a 23-mer oligonucleotide primer, and segment of about 700-base-pair was amplified only in L. monocytogenes strains and this band was visualized by hybridization with the non-radioactive probe.

Base Sequence↗
PubMed2001

PCR-based RNA probes: a quick and sensitive method to improve whole mount embryo in situ hybridizations.

We have developed a PCR-based technique for the preparation of RNA probes that can be used for whole mount in situ hybridization on embryos. T3 or T7 RNA polymerase promoters were introduced at the 5' end of gene-specific oligonucleotide primers enabling direct in vitro transcription of purified PCR fragments. We show for various marker genes in Xenopus embryos that this method provides equivalent results as compared to conventional vector-based probe preparation, even when fluorescence detection (FISH) is applied. This method offers a rapid and useful means to prepare gene-specific in situ probes predominantly for expression screens or detection of splice variants that previously required time-consuming cloning steps.

Animals↗
PubMed1995

In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.

We have developed an easy, stream-lined yet sensitive protocol for in situ hybridization to mRNA in frozen tissue sections or cytospins using digoxigenin-labeled RNA probes detected by alkaline phosphatase. We found the crucial parameters for successfully performing this technique to be tissue quality, fixation time and effective removal of excess probe. Most preparation, incubation steps and washes as described in the literature were found to be unnecessary and, therefore, eliminated, making this protocol simple enough to be achieved in any laboratory with access to tissue preparation and some molecular biology expertise.

Alkaline Phosphatase↗
PubMed1989

Human papillomavirus 6/11 and 16/18 in Schneiderian inverted papillomas. In situ hybridization with human papillomavirus RNA probes.

Schneiderian inverted papillomas may be troublesome lesions for clinicians with propensity for recurrences. Dysplasia is not uncommonly seen, and some of these lesions do progress to develop squamous carcinoma. The authors hybridized in situ seven inverted papillomas with RNA probes to human papillomavirus (HPV) 6, 11, 16, and 18. Four of these contained dysplasia, two were without dysplasia, and one contained invasive squamous cell carcinoma. Five inverted papillomas showed evidence of HPV infection based on hybridization. One with mild to moderate and one with severe dysplasia, and one without dysplasia hybridized with mixed probe HPV 6/11. One with mild dysplasia and one associated with invasive squamous cell carcinoma hybridized with mixed probe HPV 16/18. One inverted papilloma without dysplasia and one with severe dysplasia did not definitively hybridize with either mixed probe. These findings raise interesting questions as to role of HPV 6/11 and 16/18 in the development of inverted papillomas, and probably on the progression to and carcinoma.

Adult↗
PubMed1994

In-tube cDNA cloning method using a biotinylated RNA probe.

We report a simple, practical method for isolating a particular cDNA from a single-stranded (ss) cDNA library in a tube without using a radioisotope. The method consists of three steps: (i) the capture of a target cDNA by a biotinylated RNA via intermolecular hybrid formation, (ii) the binding of the cDNA-RNA hybrid to an avidin-coated gel, (iii) the recovery of the target cDNA by degradation of the RNA under mild alkaline conditions. The effectiveness of the method was examined by isolating a clone carrying a metapyrocatechase gene from a model library. This model experiment achieved an enrichment of 4800-fold. This method was applied to cloning of a cDNA encoding interleukin 8 (IL-8) from a cDNA library prepared from phorbol myristate acetate-treated U937 cells. Using an RNA probe prepared from a truncated cDNA encoding IL-8, the corresponding full-length cDNA clones were successfully obtained.

Biotin↗
PubMed1988

Colony hybridizations using nylon membranes and RNA probes--an improved method for screening bacterial clones containing bluetongue virus genome.

Bluetongue virus (BTV) total genomic and isolated individual segment dsRNAs end-labeled with 32P were successfully used as probes in colony hybridization to detect clones of BTV genomic material. The RNA probes were highly specific for cloned BTV genomic material. DNA probes, however, gave false positive results. DNA from bacterial clones was fixed to nylon and nitrocellulose membranes. The hybridized nylon membranes could be stripped of probe and reprobed at least 6 times without loss of signal strength.

Animals↗
PubMed2001

Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes.

Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2-4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68 degrees C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single-copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.

Animals↗
PubMed2000

Two-color double-labeling in situ hybridization of whole-mount Hydra using RNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization.

The freshwater polyp Hydra magnipapillata has a primitive nervous system that produces at least three distinct classes of neuropeptides: various peptides having the C-terminal sequence Arg-Phe-NH2 (the Hydra-RFamide family), Leu-Trp-NH2 (the Hydra-LWamide family), and a single peptide having the C-terminal sequence Lys-Val-NH2 (Hydra-KVamide). The various Hydra-RFamides are synthesized by three different preprohormones: preprohormone-A, -B, and -C. The various Hydra-LWamides are synthesized by a single preprohormone (prepro-Hydra-LWamide), as is Hydra-KVamide (prepro-Hydra-KVamide). Using a wholemount double-labeling two-color in situ hybridization technique and RNA probes specific for each of these five Hydra preprohormone mRNAs, we found that specific sets of neurons express each of the five preprohormones, except for the peduncle region of Hydra (an area just above the basal disk), where a population of neurons exists that expresses both preprohormones-A and preproHydra-KVamide mRNAs. The functional significance of this coexpression is unclear. This is the first report on the coexpression of two well-characterized preprohormones (yielding two well-characterized neurohormone families) in cnidarians. This report also shows that there are at least six neurochemically different populations of neurons in Hydra.

Animals↗
PubMed1985

Detection of preprocholecystokinin and preproenkephalin A mRNAs in rat brain by hybridization histochemistry using complementary RNA probes.

The distributions of mRNAs encoding preprocholecystokinin and preproenkephalin A in the rat brain were examined by in situ hybridization histochemistry. Complementary RNA probes 0.5-1.0 kilobase in length were synthesized in vitro with 35S-ribonucleotides using SP6 polymerase and a transcription vector containing the SP6 promotor. Following hybridization to fixed tissue sections, signals were detected by autoradiography after relatively short exposure times. These studies demonstrate that neuropeptide mRNAs can be detected within specific cells in the brain and that the patterns of hybridization match the localization of the peptides visualized by immunohistochemistry. Minimum numbers of mRNA copies encoding the peptides were determined by quantitative autoradiography.

Animals↗
PubMed1991

In situ hybridization using single-stranded antisense RNA probes in oral pathology research.

In situ hybridization is a new molecular biological technique which has made it possible to demonstrate messenger RNA (mRNA) transcription of a specific gene in tissue sections and in cell preparations. In addition to the immunocytochemical methods widely used to locate gene products, e.g. a protein, nucleic acid hybridization techniques are having growing impact in pathological research. Increasing availability of cloned nucleotide sequences of human genes and the recent development of very sensitive and specific hybridization methods is rapidly expanding the field of gene function studies in cell and tumour biology. As with immunocytochemical techniques, hybridization techniques require proper controls for specificity. A basic knowledge of molecular biology is essential to determine the optimal conditions for successful hybridization and to interpret the results correctly.

Antisense Elements (Genetics)↗
PubMed1995

Sensitive northern blot hybridization using digoxigenin RNA probes for the mRNA detection of two glucose transporter isoforms.

Diseases involving glucose metabolism disorders are more and more prevalent. Therefore the question of glucose transporter gene expression is being addressed in experimental and clinical studies. Radioactive probes are generally used to assess glucose transporter mRNA levels, but these probes are short-lived, costly and harmful to the environment. Alternative methods that do not present these disadvantages, for example digoxigenin (DIG) labelled probes, might prove to be very interesting for the study of glucose transporter mRNA. The aim of the present work was to compare DIG-labelled cRNA probes to 32P-labelled cRNA probes in order to see whether or not the non-radioactive method can be used to assess glucose transporter gene expression. This work shows that DIG-labelled glucose transporter (GLUT1 and GLUT4) cRNAs are suitable probes for the assessment of these gene expressions. We have found that the DIG system offers a much higher sensitivity than the 32P system for both GLUT1 and GLUT4 mRNA detection. This represents a decisive advantage in human studies where tissue quantity is a limiting factor. In addition, stability, safety, time saving and cost reduction are other considerations that make DIG-labelled GLUT1 and GLUT4 cRNAs very attractive.

Animals↗