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At least 307 records · Page 17Linked to original sources
PubMed1993

In situ hybridization of virulent canine distemper virus in brain tissue, using digoxigenin-labeled probes.

Only a few hybridization experiments have been performed for detection of canine distemper virus (CDV) nucleic acid sequences in tissue cultures and in various tissues. Those published studies used probes derived from tissue culture-adapted CDV, and hybridization signals were not obtained in the CNS tissue, although infective CDV and viral antigen were detectable in this tissue. We developed probes complementary to virulent CDV and were able to detect viral RNA not only in primary brain cell cultures, but also in brain tissues, by use of in situ hybridization. Sensitivity of the test at least equaled that of immunohistochemistry. We applied digoxigenin-labeled, strand-specific RNA probes complementary to the nucleoprotein-coding viral nucleic acid sequence. Our results indicate that to detect CDV nucleic acid sequences in brain tissues, it is essential to use probes derived from the virulent virus.

Animals↗
PubMed1995

The use of digoxigenin-labelled probes to detect DNA sequences specific for plant pathogenic bacteria.

Southern blot hybridization is a valuable method in the assessment of the pathogenicity of bacterial strains or isolates. It is also a powerful tool for the demonstration of the presence of foreign DNA sequences in the genome of genetically-engineered plant cells. In this respect, cold, digoxigenin-labelled DNA probes can be used in place of classical radioactive probes, whether hybridizations are performed on bacterial genomic or plasmidic DNA, or on plant genomic DNA. The versatility of this cold labelling makes it suitable for the detection of unique bacterial genomic or plasmid sequences, even though these are located on large plasmids. The sensitivity of this cold probe technique also permits the detection of subpicogram quantities of DNA in plant genomic preparations. Their long term storage stability allows them to be frequently re-used over long periods of time, making this technique quite cost efficient.

Bacteria↗
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↗
PubMed1995

Use of digoxigenin-labelled probes for the detection of B19 parvovirus DNA in batches of blood products.

Being thermostable and non lipid-enveloped, the B19 parvovirus cannot be destroyed by the chemical and physical treatments presently used to inactivate lipid-enveloped viruses such as HIV, hepatitis C and B viruses in plasma derivatives. A polymerase chain reaction assay was used to detect B19 parvovirus DNA. Amplified products were detected through a non-radioactive technology based on the use of a digoxigenin-labelled probe. B19 parvovirus DNA was detected in 20% of the studied batches of factor coagulation concentrates and in none of the albumin batches.

Blood↗
PubMedDate not supplied

In situ detection of reovirus in formalin-fixed, paraffin-embedded chicken tissues using a digoxigenin-labeled cDNA probe.

An in situ hybridization (ISH) technique using a digoxigenin (DIG)-labeled cDNA probe detected avian reovirus (ARV) RNA in formalin-fixed, paraffin-embedded chicken tissues. Tissues were collected 3 and 10 days following inoculation with the R-2 or the S1133 strain of ARV. The cDNA clone HJp1, located on the S1 gene segment of the ARV S1133 strain, was used to prepare a nonradioactive probe. The ISH assay localized ARV RNA in infected tissues including heart, liver, intestine, pancreas, and tendon. No positive-stained cells occurred in sections from uninfected chickens.

Animals↗
PubMed1994

Detection and identification of human papillomavirus DNA in genital tract by digoxigenin labelling using the polymerase chain reaction method.

We propose here a simple and rapid method of Human Papillomavirus (HPV) detection applied to genital samples. The method consists of a polymerase chain reaction (PCR) of samples using a consensus primer ser (Snijders et al., 1990) with labelling by Dig-dUTP (Digoxigenin deoxyuridine triphosphate) during amplification. Type identification was carried out by hybridization of the labelling PCR product with a panel of different plasmidic HPVs dotted beforehand on nylon membranes. Forty-five genital samples were tested with this method (30 samples with evident signs of HPV infection, and 15 cervical scrapes which had a normal colposcopic examination and no cytologic signs of HPV infection). We found good sensitivity and specificity levels. The labelling method during PCR did not modify the sensitivity of PCR. Forty-five genital samples were analyzed by this technique and compared with the results of dot-blot and two PCR methods using different primers. They showed a good correlation except in four samples (high grade cervical lesions) which seemed to show a partial deletion or rearrangement. Our method, used for the first time in HPV detection, needed just one PCR and one dot-blot, thus saving an important amount of time and also decreasing examination costs.

Journal Article↗
PubMed1997

Identification of Deletion Carriers in Duchenne/Becker Muscular Dystrophy Families Using a Digoxigenin-Labeled Quantitative Polymerase Chain Reaction Technique.

Background: A quantitative polymerase chain reaction (PCR) technique based on the incorporation of digoxigenin (DIG), and visualization of the labeled fragments for the detection of deletion carriers in Duchenne/Becker muscular dystrophy families has been developed. Methods and Results: Sixty-five DNA samples taken from mothers and/or sisters of familial and sporadic deletion patients were investigated in the exponential phase of amplification. All obligate carriers were correctly identified using this technique. In more than 95% of deletion families, possible carriers could be screened by using four different multiplex systems specifically designed to increase the efficiency of the detection. Deletions were found to be present in 42% of possible carriers. All these results were confirmed by computer-assisted laser densitometry. Conclusions: Dosage analysis by DIG-labeled quantitative PCR is a reliable and accurate technique for detecting Duchenne/Becker muscular dystrophy deletion carriers.

Journal Article↗
PubMed1997

In vivo and in vitro detection of canine distemper virus nucleoprotein gene with digoxigenin-labelled RNA, double-stranded DNA probes and oligonucleotides by in situ hybridization.

A single-stranded RNA, two double-stranded (ds) DNA probes and 10 oligonucleotides labelled with digoxigenin were comparatively evaluated for their usefulness to detect canine distemper virus (CDV) nucleoprotein RNA in in vitro infected Vero cells and in tissues of dogs with spontaneous CDV infection by in situ hybridization (ISH). In addition, results were compared to CDV nucleoprotein antigen distribution as demonstrated by immunohistochemistry. The RNA probe was derived from the virulent A75/17 strain, the DNA and oligonucleotide probes from the avirulent Onderstepoort strain of CDV. The two DNA probes were 287 and 126 base pairs long. For ISH, various factors including fixatives, proteolytic digestion, probe concentration, hybridization conditions and detection systems were compared. All probes were suitable for demonstration of CDV RNA in in vitro infected cells, regardless of the CDV strain employed. In vivo CDV nucleic acid was detected by RNA and the dsDNA probes. However, the probes varied substantially with respect to sensitivity and specificity. The CDV RNA probe was far superior in sensitivity when compared to the DNA probes. Furthermore, the shorter DNA probe displayed a higher sensitivity, indicating that length of the probe is an important parameter when selecting probes. Oligonucleotides displayed only rarely a positive signal and caused frequently hybridization signals in the nucleus, which where considered not specific for CDV. Summarized, the present study reveals that RNA probes are currently the most sensitive tool for detection of CDV RNA in tissues.

Animals↗
PubMed2004

Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel G protein-coupled receptor (Gpr54) during maturation in cichlid fish.

GPR54 is a novel G protein-coupled receptor speculated to be essential for sexual development. However, its role in the regulation of GnRH types is unknown. To address this issue, we cloned GPR54 from the brain of a cichlid fish (tilapia Oreochromis niloticus) and determined its expression in immature and mature males using our newly developed technique: laser-captured microdissection of single digoxigenin-labeled GnRH neurons coupled with real-time quantitative PCR. The tilapia GPR54 cDNA contains an open reading frame of 1131 bp encoding 377 amino acids and exhibits 56% identity to human GPR54. Absolute copies of GnRH1 and GnRH3, not GnRH2, mRNAs were significantly high in mature compared with immature males. At the single-cell level, only in mature males, GnRH1 mRNA levels were inversely related to GPR54 mRNA (P < 0.002). GPR54 was expressed in a significantly high percentage (45.0-60.0%) of mature GnRH1, GnRH2, and GnRH3 neurons and in immature GnRH3 neurons, which had migrated to the vicinity of their final locations in the brain; on the contrary, only 5.0% of immature GnRH1 and GnRH2 neurons had GPR54 transcripts (P < 0.001). Thus, using a novel innovative single-cell gene profiling technique, we provide evidence of the structure of a nonmammalian GPR54, which is highly conserved during evolution and is expressed in GnRH1, GnRH2, and GnRH3 neurons. Furthermore, we propose that the expression of GPR54 is a "stop signal" for GnRH1, GnRH2, and GnRH3 neuronal migration, leading to suppression of cell growth and modulation of GnRH secretion, which is important for normal sexual development.

Amino Acid Sequence↗
PubMed1999

[Detection of human papillomavirus in laryngeal carcinoma with digoxigenin labelled probe prepared by polymerase chain reaction].

OBJECTIVE: To study the relationship between laryngeal carcinoma and human papillomavirus (HPV), and describe distribution, expression of genome types of HPV in laryngeal carcinoma. METHODS: We used a non-radioactive digoxigenin labeled probe prepared by polymerase chain reaction (PCR) to test the fresh tissue samples from 146 cases with different laryngeal lesions. The labelled probe of HPV consensus primers was obtained, which enabled detection of nine types of HPV DNAs that were HPV 6, 11, 16, 18, 31, 33, 35, 42 and 58. The genome types of HPV DNA positive cases were determined by using multiple primers PCR. RESULTS: The results turned out to be as follows: The HPV infection positive rate was 45.6%(31/68) for laryngeal carcinoma, 20.0% (3/15) for neck metastatic lymphnode, 11.8%(2/17) for precarcinomatous lesions and 6.3%(1/16) for vocal cord polypus. 15 cases of normal tissues adjacent to carcinoma and another 15 normal tissues opposite to the carcinoma were all HPV DNA negative. HPV16, 18 types are the main types of laryngeal carcinoma and HPV6, 11 types often appear in benign lesions of larynx. CONCLUSION: These results implicated that the occurrence and development of laryngeal carcinoma may be closely related to HPV infection.

DNA Probes, HPV↗
PubMed1992

Optimization of non-isotopic in situ hybridization on formalin-fixed, paraffin-embedded material using digoxigenin-labelled probes and transgenic tissues.

The sensitivity of non-isotopic in situ hybridization (NISH), particularly on formalin-fixed, paraffin-embedded (FFPE) clinical tissues, has been the subject of controversy. Generally, NISH has been regarded as being less sensitive than radiolabelled procedures, although some reports have contradicted this. Accordingly, tissues from mice which were transgenic for variable amounts of the human alpha-1-antitrypsin gene were used to optimize the NISH procedure and to estimate the sensitivity. This approach showed that prolonged incubation of slides in final substrate resulted in high sensitivity--about 13 kb of target DNA. However, this prolonged incubation crucially depended on achieving minimal non-specific background staining. Many factors affected the degree of background staining, but five were particularly important. First, the method of mounting cut sections onto slides. Second, the length of the probe (ideally less than 400 bp). Third, the procedure for proteolytic digestion. Fourth, the denaturation technique, and fifth, the quality of the dextran sulphate used in the hybridization mix. The optimized protocol showed variable patterns of mRNA distribution in the transgenic mouse livers, while DNA distribution appeared uniform.

Animals↗
PubMed2001

In situ detection of messenger RNA using digoxigenin-labeled oligonucleotides and rolling circle amplification.

The detection of specific RNA molecules in situ is routinely performed using haptenated probes, which are detected by either enzymatic amplification or direct fluorescence. A drawback of fluorescence labeling has been the reduced sensitivity relative to that of methods that use enzymes as signal generators. Reliable fluorescence detection methods often require the use of multiple oligonucleotide probes for each gene target. Here, we demonstrate that single haptenated DNA probes specific for actin mRNA may be detected in situ using antibody-coupled rolling circle amplification (immuno-RCA). This fluorescence-based detection method offers remarkable sensitivity due to the use of signal amplification and yet retains the ability to count hybridization signals as discrete objects. We demonstrate the detection of actin-specific immuno-RCA signals in the cytoplasm and use 3D image deconvolution of multiple z axis sections to show that there are hundreds of signals per cell. With some modifications, this method may be adaptable to the simultaneous detection of several RNA species, including low-copy-number mRNA.

5' Untranslated Regions↗
PubMed1979

Dehydro-digitoxosides of digitoxigenin and digoxigenin: binding to beef heart (Na+ + K+)-ATPase in relation to unchanged digitoxosides.

Dehydro-digitoxosides are metabolites of digitalis glycosides. In order to study their possible biological activity their affinity to (Na+ + K+)-activated ATPase was determined and compared with unchanged glycosides. Based on the dissociation constants of glycoside-enzyme-complexes, the affinity of the dehydro-digitoxosides ranged in the same order of magnitude as that of the native glycosides. Comparing mono-, bis-, and tris-digitoxosides of digitoxigenin (dt-1, dt-2, dt-3) and of digoxin (dg-1, dg-2, dg-3) with the corresponding dehydrodigitoxosides (3'-dehydro-dt-1, 9'-dehydro-dt-2, 15'-dehydro-dt3, 3'-dehydro-dg-1 and 9'-dehydro-dg-2, respectively) the dehydro-digitoxosides had lower affinities to the enzyme. The highest dissociation constants (KD) were found for 3'-dehydro-dt-1 and 3'-dehydro-dg-1. The half maximal inhibition of (Na+ + K+)-ATPase activity (I50) coresponded to affinity measurements in all but two cases: dehydro-dt-3 and dehydro-dt-2 showed very low I50 values.

Animals↗