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At least 37 records · Page 2Linked to original sources
PubMed1993

Detection of human papillomavirus infection by non-isotopic in situ hybridisation in condylomatous and CIN lesions.

AIMS: To study the value of non-isotopic in situ hybridisation (NISH) in detecting human papillomavirus (HPV) infection in female genital lesions positive for the virus by conventional histology but negative by filter DNA hybridisation. METHODS: Forty three cases, which showed the histological hallmarks of the HPV infection but produced negative results in filter dot blot hybridisation tests (Vira Pap and Vira Type kits), were identified in the course of an investigation of 304 vaginal, vulvar, and cervical samples from 267 patients. These cases were studied by NISH for the presence of HPV infection. RESULTS: In 28 (65%) of the cases NISH gave a positive hybridisation signal. In 26 cases (96%) the signal was diffuse, and in two (4%) punctate or diffuse, representing episomal and episomal or integrated HPV DNA, respectively. In most cases only a few HPV positive cells were discernible. CONCLUSION: NISH is a more sensitive technique than dot blot hybridisation, detecting HPV infection in most cases which show histological HPV atypia but which remain negative in filter DNA hybridisation. Thus NISH is useful as an additional technique to verify the presence of HPV in lesions which remain negative in filter hybridisation tests.

Adolescent↗
PubMed1989

Detection of Y-bearing spermatozoa by DNA-DNA in situ hybridisation.

In situ hybridisation of a Y chromosome-specific DNA probe to preparations of decondensed spermatozoa revealed approximately 46.7% labelled spermatozoa among 3,900 scored. This is not significantly different from the 50% expected if only the Y chromosome-bearing spermatozoa are hybridised. Control hybridizations of Escherichia coli DNA and salmon testis DNA to decondensed sperm produced no significant labelling, whereas more than 99% of the spermatozoa were heavily labelled after hybridisation to total human DNA. These controls indicate that the methodology described in this paper renders the chromatin accessible for hybridisation and that the 50% hybridisation observed with the Y chromosome DNA probe was specific. In situ hybridisation with the Y probe therefore identifies the Y-bearing spermatozoa, and the protocol described should prove useful in evaluating methods of separating Y-bearing and X-bearing spermatozoa.

Autoradiography↗
PubMed1994

Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation.

The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.

Blastocyst↗
PubMed1975

Influence of temperature on the detectibility and chromosomal distribution of specific DNA sequences by in situ hybridisation.

Hybridising certain AT-rich satellite complementary RNAs (cRNAs) to their homologous chromosomal DNA sequences at different temperatures of incubation results in a different dispersion of autoradiographic label throughout the karyotypes. The temperature at which most label, or cRNA-DNA hybrid formation, exists corresponds to the optimal rate temperature for the hybridisation of these same satellite cRNA-DNA hybrids as determined by RNA excess filter hybridisation. It is likely that the in situ hybridisation results can therefore be explained by the fact that there is a similar temperature-dependence on the rate of hybrid formation for both in situ and RNA excess hybridisation. This should have important implications for the designing of in situ hybridisation experiments in general.

Animals↗
PubMed1979

The effects of mercury-substitution on the hybridisation characteristics of nucleic acids.

The effect of different levels of mercury substitution on the rate and extent of hybridisation of globin mRNA with a complementary DNA (cDNA) copy has been investigated. It was found that mercuration significantly reduces both the rate of hybridisation and the extent of the reaction, but that these effects are abolished when at least stoichiometric amounts of 2-mercaptoethanol are included in the hybridisation medium. As a preliminary to using this technique to isolate specific groups of sequences after long-term hybridisations, we have investigated both the rate of demercuration of RNA and its retention on thiol-sepharose columns after extended incubation under commonly employed hybridisation conditions at 43 degrees or 60 degrees. Retention was essentially quantitative even after incubation times of 300 hours at 43 degrees, but decreased significantly after 48 hours at 60 degrees. It is concluded that thiol-sepharose chromatography offers considerable advantages over hydroxyapatite chromatography for the recovery of hybridised sequences, particularly with regard to the lower levels of non-specific binding obtained and its ability to distinguish directly between DNA-DNA and DNA-Hg RNA hybrids.

Animals↗
PubMed1975

Mouse immunoglobulin genes: studies on the reiteration frequency of light-chain genes by hybridisation procedures.

The partially purified immunoglobulin light chain messenger RNA fraction from P3K (MOPC 21) mouse myeloma tissue-culture cells has been employed in hybridisation studies. Fragments of the messenger RNA were generated by alkali hydrolysis. 6-S fragments not containing poly(A) showed the characteristic biphasic hybridisation profile seen with the intact RNA fraction. 12-S and 6-S poly(A)-containing fragments, however, showed single transitions lacking the rapidly hybridising component. Complementary DNA copies of the intact messenger RNA fraction were prepared with RNA-dependent DNA polymerase and the DNA populations fractionated on acrylamide gels. Hybridisation experiments with complementary DNA fractions up to 800 bases in length showed annealing to single (or a few) genes. A rapidly hybridising component (about 200 copies) appears in the cDNA fraction containing the largest transcripts. We conclude that the kappa constant region gene and the MOPC 21 variable region gene are present as one or a few copies in the haploid genome and that the rapidly hybridising component is not due to variable region genes.

Animals↗
PubMed1989

Detection of Campylobacter pylori in stomach tissue by DNA in situ hybridisation.

A non-radioactive DNA in situ hybridisation (DISH) protocol was developed. It requires the use of biotinylated Campylobacter pylori DNA as the probe to detect C pylori DNA in routinely embedded stomach biopsy specimens. In sequential tissue samples from a 58 year old woman with recurrent chronic active gastritis the C pylori probe hybridised with bacteria whenever they were histologically visible. When no bacteria were visible histologically, hybridisation was negative with one exception. In a single biopsy specimen without visible C pylori, hybridisation occurred with the surface of the antrum epithelium, while control hybridisation with a heterologous probe remained negative. From a parallel biopsy specimen taken at the same time the C pylori culture was positive. It is concluded that DISH, although more laborious than routine staining techniques, may be more sensitive in that it detects bacteria very easily, even when sections are not counterstained or when they are present in low numbers, and that bacteria which do hybridise are unequivocally identified as C pylori and not Campylobacter-like organisms.

Campylobacter↗
PubMed1990

In situ hybridisation in herpetic lesions using a biotinylated DNA probe.

In situ hybridisation was performed with a biotinylated DNA probe for herpes simplex virus (HSV) using high temperature denaturation on formalin fixed, paraffin wax sections of lung, brain, ganglion and keratinising and non-keratinising squamous epithelia. Eosinophilic viral nuclear inclusions or characteristically moulded multiple nuclei with altered chromatin, which were present in two cases of HSV encephalitis and one case of viral pneumonitis, all showed complete hybridisation visualised by an alkaline phosphatase/nitroblue tetrazolium detector system. HSV encephalitis and trigeminal ganglionitis, which were confirmed serologically or clinicopathologically but lacked nuclear changes, also gave positive dense nuclear signal in neurons, glias and satellite cells. No staining was present in the ganglion cells in trigeminal zoster, the glia in progressive multifocal leucoencephalopathy, or in a variety of cells in a lung coinfected with cytomegalovirus. In 10 herpetic blisters of squamous epithelia, infected cells hybridised strongly, while morphologically similar herpes zoster lesions remained negative. In neural tissues non-hybridisation staining was most obtrusive in corpora amylacea and seemed to reflect nonspecific probe adherence. In squamous epithelium, major non-hybridisation staining was caused by probe and antibody possibly adhering to intracellular keratin. The HSV probe permits specific detection of virus in the absence of characteristic nuclear changes and allows varicella zoster virus to be differentiated from HSV, provided that the aforementioned problems with non-hybridisation staining are borne in mind.

Biotin↗
PubMed1998

Detection of cytomegalovirus in upper gastrointestinal biopsies from heart transplant recipients: comparison of light microscopy, immunocytochemistry, in situ hybridisation, and nested PCR.

AIM: To establish the diagnostic value of in situ hybridisation and the nested polymerase chain reaction (PCR) in detecting clinically relevant cytomegalovirus (CMV) infection in upper gastrointestinal biopsies from heart transplant patients. METHODS: Test sensitivity and specificity for detection of CMV early gene RNA by in situ hybridisation and CMV intermediate early gene by PCR were established and then compared with haematoxylin and eosin (H&E) and immunocytochemical detection of CMV in order to establish the best pathological diagnostic approach. All investigations were carried out on formalin fixed, paraffin embedded tissue. RESULTS: Nested PCR had the highest test sensitivity, followed by in situ hybridisation and immunocytochemistry with the same sensitivity; H&E had the lowest. H&E and immunocytochemistry were the most specific but both had a significant false negative rate which was less of a problem with PCR. However, PCR gave no other diagnostic information, and in situ hybridisation was no better than immunocytochemistry. Both in situ hybridisation and PCR were technically complex and more expensive. CONCLUSIONS: H&E and immunocytochemistry represent the best initial screen for CMV and other diseases in upper gastrointestinal biopsies from heart transplant patients. If H&E and immunocytochemistry were negative, nested PCR could significantly increase the diagnostic yield of clinically relevant CMV infection. In situ hybridisation appeared to have no advantages and some drawbacks compared with immunocytochemistry and PCR.

Adult↗
PubMed1997

Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide.

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embedded sections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes.

Biotin↗
PubMed1991

Detection of B19 parvovirus in human fetal tissues by in situ hybridisation.

Evidence of B19 parvovirus infection was sought by in situ hybridisation with biotinylated probes in 65 tissue samples from 32 pregnancies (fetuses, products of conception and/or placentas). Twenty-seven samples were reactive and the results were confirmed by other methods for B19 virus detection in 22 cases. The other methods used were in situ hybridisation with 3H and 35S labelled probes; dot-blot hybridisation with biotin and 32P labelled probes; polymerase chain reaction assay; negative stain and thin section electron microscopy; and radioimmunoassay for B19 antigen. The five false positive results by in situ hybridisation with biotinylated probes were considered to be due to non-specific biotin capture and were more frequent with unfixed samples than with formalin fixed material. It was concluded that while biotinylated probes offered advantages over radioactive probes for detecting B19 DNA by in situ hybridisation, positive findings should be confirmed by other methods.

Biotin↗
PubMed1993

Comparison of 3' and 5' biotin labelled oligonucleotides for in situ hybridisation.

Oligonucleotide probes enzymatically labelled at the 3'-end with biotin have been used successfully to detect target RNA and DNA in combination with in situ hybridisation. Addition of multiple biotin residues to the 3'-end increases the hybridisation signals, but it is not known whether the same principle is applicable to the 5'-end. We have labelled a 35-base oligonucleotide during synthesis with 1, 5 and 12 biotin molecules at the 5'-end and compared it to conventional 3'-labelling. In additional experiments the probes were labelled at both ends. Probes were applied to histological sections obtained from paraffin-embedded cell-clot-complexes that contain uninfected and Rhinoviral-infected cells, using a standard in situ hybridisation protocol with appropriate controls. Hybridisation signals were compared for intensity of cytoplasmic signal and sensitivity as number of positive cells. Both parameters increased in parallel with higher numbers of biotin residues attached to the 5'-end and 12 biotin residues were almost as effective as 3'-enzymatic tailing. The sensitivity could be increased above that of either 3'- or 5'-labelling by the addition of residues at both ends of the probe. The 5'-attachment of biotin residues can extend the value of oligonucleotide probes employed for in situ hybridisation and yield increased sensitivity when combined with 3'-enzymatic labelling.

Animals↗
PubMed1988

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature, pubertal, pregnant, lactating and ovariectomised rats.

We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a 32P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.

Animals↗
PubMed1999

A comparison of six cypovirus isolates by cross-hybridisation of their dsRNA genome segments.

Genetic relationships between the genome segments of six cypovirus (CPV) isolates were analysed by RNA cross-hybridisation. These included three type 1 viruses and single isolates of types 2, 5 and 12, which collectively are identical to those previously compared by serology and electrophoresis [Mertens et al. (1989), J Gen Virol 70: 173-185]. Since only genome segment 10 of three cypovirus types and segments 8 and 9 of a single virus strain (of type 1) have currently been sequenced, this initial study provides some additional information on sequence variation/similarity in each of the ten genome segments. The RNA of the type 1 viruses showed high levels of cross-hybridisation. Significant but much lower levels of cross-hybridisation were detected between type 1 and the related type 12 CPV. However, only very low levels of cross-hybridisation were detected between the other pairs of viruses. Apart from evidence of a slightly higher level of sequence similarity between the largest segments, the RNA sequence appeared to vary uniformly across the whole genome. There was no evidence for any type specific RNA sequences restricted to individual genome segment(s). The sequence variation, reflected in the levels of RNA sequence similarity and cross hybridisation, correlates well with serological data, showing large differences between CPV types and supports the continued use of electropherotype as one of the 'species parameters' for the classification of cypoviruses.

Animals↗
PubMed1991

Sensitive non-isotopic DNA hybridisation assay or immediate-early antigen detection for rapid identification of human cytomegalovirus in urine.

A sensitive non-radioactive DNA hybridisation assay employing digoxigenin-labelled probes was compared with immediate-early antigen detection and conventional virus isolation for the identification of human cytomegalovirus (HCMV) in 249 urine samples. Of 44 specimens yielding HCMV by virus isolation, more were positive by DNA hybridisation (32; 73%) than by immediate-early antigen detection (25; 52%) (P = 0.05). The specificity of the hybridisation assay in 45 apparently falsely positive specimens was supported by detection of HCMV DNA in 40 of these specimens using the polymerase chain reaction. Many urine specimens may thus contain large amounts of non-viable virus or free viral DNA. Evaluation of various protocols for the extraction and denaturation of virus DNA prior to hybridisation showed that proteinase K digestion with phenol/chloroform extraction was the most sensitive and reliable procedure. We conclude that the non-radioactive DNA hybridisation assay described is a potentially valuable routine diagnostic test.

Antigens, Viral↗
PubMed1984

Novel identification of differences in the kinetoplast DNA of Leishmania isolates by recombinant DNA techniques and in situ hybridisation.

Kinetoplast DNA (kDNA) was extracted from marker isolates of three 'Old World' cutaneous leishmania, L. tropica, L. aethiopica and L. major. Restriction endonuclease digestion followed by electrophoresis showed that each isolate produced a unique pattern of DNA fragments. The kDNA of each isolate was hybridised to Southern blots of digests of all three kDNAs. This showed that the kDNA of each isolate contained sequences unique to that isolate as well as sequences common to all three isolates. The kDNA sequences of L. tropica and L. aethiopica were more closely related than either were to those of L. major. kDNA of each isolate was cloned and plasmids selected which contained a fragment of DNA unique to the isolate from which the kDNA originated. Whole kDNA was hybridised to organisms fixed on a microscope slide. In each case the kDNA hybridised strongly to the kinetoplast of the organism from which the DNA had been extracted. There was a small amount of cross-hybridisation between L. tropica and L. aethiopica confirming the result of the Southern blot hybridisation. The feasibility of a method of isolate identification using recombinant DNA probes containing sequences unique to a particular isolate or group of isolates is discussed.

Animals↗
PubMed1994

Relational genome analysis using reference libraries and hybridisation fingerprinting.

The genomes of eukaryotic organisms are studied by an integrated approach based on hybridisation techniques. For this purpose, a reference library system has been set up, with a wide range of clone libraries made accessible to probe hybridisation as high density filter grids. Many different library types made from a variety of organisms can thus be analysed in a highly parallel process; hence, the amount of work per individual clone is minimised. In addition, information produced on one analysis level instantly assists in the characterisation process on another level. Genetic, physical and transcriptional mapping information and partial sequencing data are obtained for the individual library clones and are cross-referenced toward a comprehensive molecular understanding of genome structure and organisation, of encoded functions and their regulation. The order of genomic clones is established by hybridisation fingerprinting procedures. On these physical maps, the location of transcripts is determined. Complementary, partial sequence information is produced from corresponding cDNAs by hybridising short oligonucleotides, which will lead to the identification of regions of sequence conservation and the constitution of a gene inventory. The hybridisation analysis of the cDNA clones, and the genomic clones as well, could potentially be expanded toward a determination of (nearly) the complete sequence. The accumulated data set will provide the means to direct large-scale sequencing of the DNA, or might even make the sequence analysis of large genomic regions a redundant undertaking due to the already collected information.

Animals↗
PubMed1995

Identified cholinergic neurones in the adult rat brain are enriched in GAP-43 mRNA: a double in situ hybridisation study.

The cellular expression of growth associated protein-43 mRNA by identified choline acetyl transferase mRNA positive cells was investigated in the mature rat brain using a combined radioactive and non-radioactive in situ hybridisation technique. Cellular sites of growth associated protein-43 mRNA were detected using a 35S-oligonucleotide while choline acetyl transferase mRNA positive neurones were identified using two alkaline phosphatase-labelled probes. In the cholinergic cells of the corpus striatum, basal forebrain and laterodorsal tegmental nucleus a specific growth associated protein-43 hybridisation signal (silver grains) was detected, demonstrating that these choline acetyl transferase mRNA positive cells are enriched in growth associated protein-43 gene transcripts. By contrast, the large cholinergic cells of the motor nucleus of the trigeminal nerve did not express growth associated protein-43 mRNA. Quantification of the growth associated protein-43 hybridisation signal expressed by identified choline acetyl transferase mRNA positive cells showed regional variations in the relative cellular abundance of this transcript; cholinergic cells in the laterodorsal tegmental nucleus and corpus striatum expressed the strongest cellular hybridisation signal. Mean cross-sectional somatic area measurements of these growth associated protein-43/cholinergic positive cells confirmed the identity of these neurones as belonging to the cholinergic phenotype. A strong 35S-growth associated protein-43 hybridisation signal was detected also in numerous other non-choline acetyl transferase mRNA positive nerve cells in other regions of the brain, although the chemical phenotypes of these neurones were not determined. Our data reveal that expression of the growth-associated protein GAP-43 is maintained in identified cholinergic neurones in the postnatal rat brain, suggesting that this protein may subserve important functions in cholinergic and other neurones of the adult mammalian brain.

Animals↗