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At least 109 records · Page 6Linked to original sources
PubMed1984

Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands.

We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.

Animals↗
PubMed1987

Localization of somatostatin mRNA in the gut, pancreas and thyroid gland of the rat using antisense RNA probes for in situ hybridization.

A method for the localization of rat pre-prosomatostatin mRNA by in situ hybridization with 32P- and 3H-labeled antisense RNA probes is reported. Somatostatin mRNA was detected in endocrine cells of the rat gut mucosa, pancreas and thyroid in a distribution identical to immunoreactive somatostatin. In addition, in situ hybridization allowed localization of reactive neurons in the submucous and myenteric plexus of the gut, sites which are variably positive or negative for immunoreactive somatostatin even after colchicine treatment. These studies indicate that in situ hybridization is more sensitive than immunohistochemistry in some instances for demonstration of somatostatin in gene expression.

Animals↗
PubMed1988

Structure and function of Y chromosomal DNA. II. Analysis of lampbrush loop associated transcripts in nuclei of primary spermatocytes of Drosophila hydei by in situ hybridization using asymmetric RNA probes of four different families of repetitive DNA.

pSP64/65 subclones of four different families of repetitive sequences on the Y chromosome of Drosophila hydei were used for in vitro synthesis of labelled RNA. Pairs of RNA probes of opposite strand polarity were employed to analyse RNAs transcribed on, or associated with, various Y chromosomal lampbrush loops in nuclei of primary spermatocytes of D. hydei. The results of RNA filter analysis and in situ hybridization experiments can be generalized as follows: (1) Y-specific transcripts are heterogeneous in length and are synthesized on lampbrush loops. (2) Transcription of tandemly repeated sequences is usually strand specific. (3) Members of the same sequence family can be found in transcripts from different lampbrush loops. (4) Transcripts not coded by the Y chromosome are accumulated on different subregions of Y chromosomal lampbrush loops.

Animals↗
PubMed1990

Typing of coagulase-negative staphylococci by Southern hybridization of chromosomal DNA fingerprints using a ribosomal RNA probe.

Ribotyping consists of restriction endonuclease fingerprinting of ribosomal RNA (rRNA) genes visualized by Southern hybridization with an rRNA probe. This method was developed and compared with restriction endonuclease fingerprinting of chromosomal DNA for typing coagulase-negative staphylococci. Twenty-five American Type Culture Collection reference type strains and 53 clinical isolates were typed. Both methods clearly distinguished all 15 species of coagulase-negative staphylococci and most individual strains within each species. Except in the case of Staphylococcus warneri, ribotyping was most discriminating with the use of ClaI, one of eight endonucleases tested. HpaI and AvaI were more specific than ClaI for discrimination between strains of Staphylococcus warneri. The patterns produced by ribotyping were much simpler and thus easier to interpret than corresponding chromosomal fingerprints. However, ribotyping was slightly less discriminating. It is concluded that ribotyping offers an alternative method for molecular typing of coagulase-negative staphylococci. The application of both methods needs to be further evaluated in the clinical setting.

Bacterial Typing Techniques↗
PubMed1995

A comparison of digoxigenin and biotin labelled DNA and RNA probes for in situ hybridization.

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes achieved maximal sensitivity without use of biotin-strep-tavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.

Animals↗
PubMed2005

Ribosomal RNA probes and microarrays: their potential use in assessing microbial biodiversity.

The awareness that global biological diversity is affected by numerous, mostly human-made threats has made biodiversity assessment an important scientific issue for decades. Biodiversity includes different levels of complexity, such as community diversity, habitat diversity, genetic diversity, and species diversity. The application of molecular methods to answer ecological questions permits issues of biodiversity to be addressed at all levels. Microorganisms dominate global biological diversity in terms of their species numbers. However, their small size and limited morphological features make it challenging to obtain a comprehensive view of their biodiversity. The application of ribosomal RNA (rRNA) probes contributes significantly to the assessment of biodiversity at the molecular level. DNA microarrays offer a great potential to facilitate the application of molecular probes and other DNA analytical methods to answer ecological and biodiversity questions. We provide an introduction into the application of rRNA probes and DNA microarrays for the assessment of microbial biodiversity, as well as protocols for the implementation of DNA microarrays.

Base Sequence↗
PubMed1998

In situ hybridization with 33P-labeled RNA probes for determination of cellular expression patterns of liver transcription factors in mouse embryos.

Murine hepatocyte nuclear factor-3beta (HNF-3beta) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation. HNF-3beta also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelium, and pancreatic acinar cells. We have previously identified a hepatocyte and pancreatic cut-homeodomain transcription factor, HNF-6, which is required for HNF-3beta promoter activity. In this study, we used in situ hybridization studies of stage-specific embryos to demonstrate that HNF-6 and its target gene, HNF-3beta, are coexpressed in the foregut endoderm and in the pancreatic and hepatic diverticulum. More detailed analysis of HNF-6 and HNF-3beta's developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelium, and pancreatic ductal epithelium and exocrine acinar cells. In support of the role of HNF-6 in regulating HNF-3beta expression in developing hepatocytes, their liver expression levels are both transiently reduced between 14 and 15 days of gestation. At day 18 of gestation and in adult pancreas, HNF-6 and HNF-3beta transcripts remain colocalized in the exocrine acinar cells, but their expression patterns diverge in endocrine cells. HNF-3beta expression is restricted to the endocrine cells of the islets of Langerhans, whereas the ductal epithelium expresses HNF-6. We discuss these expression patterns with respect to specification of hepatocytes and differentiation of the endocrine and exocrine pancreas.

Animals↗
PubMed1987

Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe.

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH beta subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LH beta cRNA probe (bio-LH beta-cRNA) was applied to detect LH beta mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LH beta-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LH beta mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LH beta mRNA. After castration (2-4 weeks) this percentage rose to 19-24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LH beta mRNA per cell, and a 2.2-fold increase in the area of cells containing LH beta mRNA. Hence, castration resulted in an increase in the level of LH beta mRNA per cell as well as the number of LH beta mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LH beta and 80% of gonadotropes that stained for FSH beta contained LH beta mRNA whereas after castration 99% of LH-storing and 93% of FSH-storing cells contained LH beta mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.

Affinity Labels↗
PubMed1991

Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts.

We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occurring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNA-DNA hybrids was observed. The apparent affinity of the antibodies to short (less than or equal to 11 bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm- values of GRV dsRNA-3.

Animals↗
PubMed1995

Isolation of viroid-RNA-binding proteins from an expression library with nonradioactive-labeled RNA probes.

The detection and isolation of cDNAs of tomato proteins that are able to bind to viroid RNA molecules are described. They were found by screening of a lambda gt11 cDNA expression library using a modification of the previously established ligand-blotting procedure to detect DNA- and RNA-binding proteins. The essentials of our modifications are the use of (i) digoxigenin-labeled viroid RNA, (ii) low concentration of the labeled probes and (iii) an expression library that allows the direct isolation of cDNA clones. The analysis of various isolated clones showed that this method is reliable for RNA-ligand screening and North-Western blotting. Applied to viroid RNA, these experimental tools provide the precondition for further studies on the specificity of the isolated proteins.

Gene Library↗