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Sample records for fluorescence in situ hybridization

  1. Fluorescence In Situ Hybridization on Rice Chromosomes.

    PubMed

    Li, Yafei; Cheng, Zhukuan

    2016-01-01

    Fluorescence in situ hybridization (FISH) has become one of the most important technologies applied in plant molecular cytogenetic research. FISH technique has been not only well applied in physical mapping and genomic studies, but also served as an indispensable tool in tracing the individual chromosome during cell division. This chapter provides protocols for basic FISH analysis using rice as a model, which can also be adapted to other model plant species. PMID:26659957

  2. Molecular cytogenetics using fluorescence in situ hybridization

    SciTech Connect

    Gray, J.W.; Kuo, Wen-Lin; Lucas, J.; Pinkel, D.; Weier, H-U.; Yu, Loh-Chung.

    1990-12-07

    Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. in this approach, termed molecular cytogenetics, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. To accomplish this, the DNA in the target cells is made single stranded by thermal denaturation and incubated with single-stranded, chemically modified probe under conditions where the probe will anneal only with DNA sequences to which it has high DNA sequence homology. The bound probe is then made visible by treatment with a fluorescent reagent such as fluorescein that binds to the chemical modification carried by the probe. The DNA to which the probe does not bind is made visible by staining with a dye such as propidium iodide that fluoresces at a wavelength different from that of the reagent used for probe visualization. We show in this report that probes are now available that make this technique useful for biological dosimetry, prenatal diagnosis and cancer biology. 31 refs., 3 figs.

  3. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 2011-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration...Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration...Controls Guidance Document: Automated Fluorescence in situ Hybridization (FISH)...

  4. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 2010-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration...Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration...Controls Guidance Document: Automated Fluorescence in situ Hybridization (FISH)...

  5. [Fluorescence in situ hybridization on histologic sections].

    PubMed

    Mrhalová, Marcela; Kodet, Roman

    2013-10-01

    I-FISH (fluorescence in situ hybridization on interphasic nuclei) represents a laboratory method linking morphological investigations (histological sections of formaldehyde fixed and paraffin embedded tissues) with molecular techniques (sequence specificity of nucleic acids bases for a certain locus). I-FISH is relatively undemanding for a laboratory workout, but offering a lot of important information about the investigated cells. Within a scope of pathology departments I-FISH is utilized mostly in diagnostics of neoplasms. I-FISH is helpful in detecting gene copy numbers (amplifications or deletions), and, importantly, in establishing copy numbers of individual chromosomes (polysomies or monosomies), chromosomal breaks and translocations. At present, I-FISH is used not only for diagnosis and estimation of prognosis, but also as a method to qualify a patient for a targeted biological therapy. Because demands on investigation of solid tumors keep raising I-FISH becomes a part of routine investigations. The aim of this paper is to summarize principles and the utility of I-FISH and to help the interested readers in finding a basic orientation in this laboratory method. PMID:24289480

  6. FISH-ing for Genes: Modeling Fluorescence "in situ" Hybridization

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck

    2006-01-01

    Teaching methods of genetic analysis such as fluorescence in situ hybridization (FISH) can be an important part of instructional units in biology, microbiology, and biotechnology. Experience, however, indicates that these topics are difficult for many students. The authors of this article describe how they created an activity that effectively…

  7. 10p Duplication characterized by fluorescence in situ hybridization

    SciTech Connect

    Wiktor, A.; Feldman, G.L.; Van Dyke, D.L.; Kratkoczki, P.; Ditmars, D.M. Jr.

    1994-09-01

    We describe a patient with severe failure to thrive, mild-moderate developmental delay, cleft lip and palate, and other anomalies. Routine cytogenetic analysis documented a de novo chromosome rearrangement involving chromosome 4, but the origin of the derived material was unknown. Using chromosome specific painting probes, the karyotype was defined as 46,XY,der(4)t(4;10)(q35;p11.23). Characterization of the dup(10p) by fluorescence in situ hybridization (FISH) analysis provides another example of the usefulness of this technology in identifying small deletions, duplications, or supernumerary marker chromosomes. 19 refs., 4 figs.

  8. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated fluorescence in situ hybridization (FISH... Laboratory Equipment and Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification. An automated FISH enumeration system is a device that consists of...

  9. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated fluorescence in situ hybridization (FISH... Laboratory Equipment and Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification. An automated FISH enumeration system is a device that consists of...

  10. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated fluorescence in situ hybridization (FISH... Laboratory Equipment and Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification. An automated FISH enumeration system is a device that consists of...

  11. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated fluorescence in situ hybridization (FISH... Laboratory Equipment and Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification. An automated FISH enumeration system is a device that consists of...

  12. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated fluorescence in situ hybridization (FISH... Laboratory Equipment and Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification. An automated FISH enumeration system is a device that consists of...

  13. Human cDNA mapping using fluorescence in situ hybridization

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  14. Characterization of Robertsonian translocations by using fluorescence in situ hybridization

    SciTech Connect

    Wolff, D.J.; Schwartz, S. )

    1992-01-01

    Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions.

  15. Detection of dengue group viruses by fluorescence in situ hybridization

    PubMed Central

    2012-01-01

    Background Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays. PMID:23110979

  16. The Application of Fluorescence In Situ Hybridization in Different Ploidy Levels Cross-Breeding of Lily

    PubMed Central

    Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

    2015-01-01

    21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid ‘Freya’ had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true. PMID:26010356

  17. The application of fluorescence in situ hybridization in different ploidy levels cross-breeding of lily.

    PubMed

    Wang, Qing; Wang, Jingmao; Zhang, Yiying; Zhang, Yue; Xu, Shunchao; Lu, Yingmin

    2015-01-01

    21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid 'Freya' had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true. PMID:26010356

  18. Role of fluorescence in situ hybridization (FISH) in sequencing the tomato genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromosomes at various stages of the cell cycle can be used for localization of DNA probes via Fluorescence in situ hybridization (FISH). While mitotic metaphase chromosomes are demonstrably too short and compact for this purpose, long pachytene chromosomes are ideal. BACs that hybridize to euchrom...

  19. Sperm Identification in Maize by Fluorescence in Situ Hybridization.

    PubMed Central

    Shi, L.; Zhu, T.; Mogensen, H. L.; Keim, P.

    1996-01-01

    The two sperm cells of common origin within the pollen tube of flowering plants are each involved in a fertilization event. It has long been recognized that preferential fusion of one sperm with the egg can occur in B chromosome-containing lines of maize. If the second pollen mitosis begins with a single B chromosome, nondisjunction will result in one sperm possessing two B chromosomes and the other containing no B chromosomes. The B chromosome-containing sperm most often fertilizes the egg, whereas the sperm nucleus with no B chromosomes fuses with the polar nuclei. Despite the obvious advantages of being able to recognize and then track, separate, and analyze one sperm type from the other, it has not been possible because of the lack of sufficient detectable differences between the two types of sperms. In this study, we used a B chromosome-specific DNA sequence (pZmBs) and in situ hybridization to identify and track the B chromosome-containing sperm cell within mature pollen and pollen tubes. Our results are consistent with conclusions from previous genetic studies related to B chromosome behavior during pollen formation. Within pollen tubes, the position in which the B chromosome-containing sperm travels (leading or trailing) in relation to the sperm cell lacking B chromosomes appears to be random. PMID:12239402

  20. SPATIAL DISTRIBUTION OF SPERM-DERIVED CHROMATIN IN ZYGOTES DETERMINED BY FLUORESCENCE IN SITU HYBRIDIZATION

    EPA Science Inventory

    Fluorescence in situ hybridization was used to determine the spatial distribution of chromatin in zygote pronuclei. ybrid system involving golden hamster eggs and individual human sperm permitted use of DNA probes specific for the entire human chromosome 4, for the heterochromati...

  1. Evolution of Chromosome 6 of Solanum Species Revealed by Comparative Fluorescence in Situ Hybridization Mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genome mapping is an important tool in evolutionary research. Here we demonstrate a comparative fluorescent in situ hybridization (FISH) mapping strategy. A set of 13 bacterial artificial chromosome (BAC) clones derived from potato chromosome 6 was used for FISH mapping in seven differen...

  2. De Novo nonreciprocal translocation 1;8 confirmed by fluorescent in situ hybridization

    SciTech Connect

    Wiley, J.E.; Stout, C.; Palmer, S.M.

    1995-07-17

    Constitutional nonreciprocal translocations are extremely rare, and even their existence is controversial. We report on a newborn infant with a de novo nonreciprocal translocation between chromosomes 1 and 8 resulting in 1q42.3 deletion syndrome. Fluorescent in situ hybridization with whole chromosome paints confirmed the conventional cytogenetic diagnosis. 3 refs., 2 figs., 1 tab.

  3. A supersandwich fluorescence in situ hybridization strategy for highly sensitive and selective mRNA imaging in tumor cells.

    PubMed

    Huang, Jin; Wang, He; Yang, Xiaohai; Yang, Yanjing; Quan, Ke; Ying, Le; Xie, Nuli; Ou, Min; Wang, Kemin

    2016-01-01

    We report a supersandwich fluorescence in situ hybridization (SFISH) strategy for highly sensitive and selective in situ visualization of mRNA expression patterns at the single-cell level. This strategy uses two fluorophore-labeled signal probes to generate a supersandwich product, which in turn generates numerous signal probes located at the target mRNA position, resulting in the in situ fluorescence signal amplification. PMID:26523451

  4. Mapping of a rat multidrug resistance gene by fluorescence in situ hybridization

    SciTech Connect

    Popescu, N.C.; Silverman, J.A.; Thorgeirsson, S.S. )

    1993-01-01

    A cDNA clone encoding the rat mdr1b (Pgy2) gene was recently isolated and characterized. This gene has a high degree of sequence identity with other Pgy genes, particularly the mouse Pgy2 gene. By means of in situ fluorescence hybridization, the rat Pgy gene was localized on chromosome 4 band q12. This regional mapping will facilitate the identification of synteny groups on rat, mouse, and human genomes and chromosomal rearrangements during mammalian evolution. 17 refs., 2 figs.

  5. Fluorescent In Situ Hybridization to Detect Transgene Integration into Plant Genomes

    NASA Astrophysics Data System (ADS)

    Schwarzacher, Trude

    Fluorescent chromosome analysis technologies have advanced our understanding of genome organization during the last 30 years and have enabled the investigation of DNA organization and structure as well as the evolution of chromosomes. Fluorescent chromosome staining allows even small chromosomes to be visualized, characterized by their composition and morphology, and counted. Aneuploidies and polyploidies can be established for species, breeding lines, and individuals, including changes occurring during hybridization or tissue culture and transformation protocols. Fluorescent in situ hybridization correlates molecular information of a DNA sequence with its physical location on chromosomes and genomes. It thus allows determination of the physical position of sequences and often is the only means to determine the abundance and distribution of DNA sequences that are difficult to map with any other molecular method or would require segregation analysis, in particular multicopy or repetitive DNA. Equally, it is often the best way to establish the incorporation of transgenes, their numbers, and physical organization along chromosomes. This chapter presents protocols for probe and chromosome preparation, fluorescent in situ hybridization, chromosome staining, and the analysis of results.

  6. Automatic Signal Classification in Fluorescence In Situ Hybridization Images

    E-print Network

    Lerner, Boaz

    an accurate and efficient alternative to that obtained using an auto-focusing mechanism. Cytometry 43. Moreover, this mechanism has to be activated for each and every field of view (FOV). However, an auto on an auto-focusing method for obtaining a clearly defined image. Because signals are distributed in three

  7. Fluorescence in situ hybridization on formalin-fixed, paraffin-embedded tissue sections.

    PubMed

    Zordan, Adrian

    2011-01-01

    Although in situ hybridization has been in use for over 30 years, its application to the study of solid tissue has only recently been adopted. Despite the numerous reports of the viability of formalin-fixed, paraffin-embedded (FFPE) tissue for fluorescence in situ hybridization (FISH) testing, this technique has not been universally implemented in the routine diagnostic setting. This is most likely due to the perception that the process is more technically demanding than FISH using conventional cytogenetic samples. FFPE FISH does, however, enable retrospective analysis of archived tissue samples and is helpful in the diagnosis of morphologically difficult cases such as Burkitt-like lymphoma, diffuse large B-cell lymphoma, and mantle-cell lymphoma. PMID:21431643

  8. Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter.

    PubMed

    Taguchi, Tomoyuki; Shinozaki, Youhei; Takeyama, Haruko; Haraguchi, Satoshi; Yoshino, Masato; Kaneko, Masao; Ishimori, Yoshio; Matsunaga, Tadashi

    2006-11-01

    This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe. PMID:16793153

  9. Multicolor fluorescent in situ mRNA hybridization (FISH) on whole mounts and sections.

    PubMed

    Lea, Robert; Bonev, Boyan; Dubaissi, Eamon; Vize, Peter D; Papalopulu, Nancy

    2012-01-01

    In situ hybridization involves the hybridization of an antisense RNA probe to an mRNA transcript and it is a powerful method for the characterization of gene expression in tissues, organs, or whole organisms. Performed as a whole mount (WISH), it allows the detection of mRNA transcripts in three dimensions, while combined with sectioning, either before or after hybridization, it provides gene expression information with cellular resolution. FISH relies on the fluorescence detection of probes and is the method of choice for the simultaneous detection of transcripts with similar or overlapping expression patterns, as each can be clearly distinguished by the selection of fluorophore. Here, we describe a protocol for performing multicolor FISH in Xenopus embryos in whole mounts and sections that can be further combined with antibody staining. PMID:22956102

  10. Physical mapping of chromosome 17 cosmids by fluorescence in situ hybridization and digital image analysis

    SciTech Connect

    Kallioniemi, O.P.; Kallioniemi, A.; Sudar, D.; Pinkel, D.; Gray, J. ); Mascio, L. ); Deaven, L. )

    1994-03-01

    The authors used fluorescence in situ hybridization and digital image analysis to localize cosmids along human chromosome 17. Seventy-one cosmids were selected at random from a chromosome 17 library constructed from a partial Sau3AI digest of flow-sorted chromosomes from a mouse-human hybrid cell line. Sixty-three of these (89%) gave a signal only on chromosome 17. The 40 cosmids producing the most distinct hybridization signals in metaphase and interphase cells were precisely mapped using digital image analysis. An additional 20 cosmids, previously mapped by linkage analysis, were also mapped. The order of these probes determined by metaphase mapping was consistent with the order determined by linkage analysis. 13 refs., 1 fig., 1 tab.

  11. Multiplexed miRNA fluorescence in situ hybridization for formalin-fixed paraffin-embedded tissues.

    PubMed

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2014-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  12. Classification of fluorescence in situ hybridization images using belief networks

    E-print Network

    Lerner, Boaz

    since it allows numerical chromosome abnormalities to be de- tected during normal cell interphase. One of a belief network are learned in order to classify images enabling the detection of genetic abnormalities of genetic abnormalities. FISH offers numerous advantages compared with con- ventional cytogenetic techniques

  13. Genomic analysis of sorghum by fluorescence in situ hybridization 

    E-print Network

    Kim, Jeong-Soon

    2004-11-15

    are likely to be broad. In the first study, I developed a FISH-based karyotyping system for Sorghum bicolor Moench. I used integrated structural genomic resources, including linkage maps and large-insert clonal libraries of sorghum genomic DNA to develop a...

  14. Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids.

    PubMed

    Fontenete, Silvia; Silvia, Fontenete; Barros, Joana; Joana, Barros; Madureira, Pedro; Pedro, Madureira; Figueiredo, Céu; Céu, Figueiredo; Wengel, Jesper; Jesper, Wengel; Azevedo, Nuno Filipe; Filipe, Azevedo Nuno

    2015-05-01

    In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work. PMID:25840566

  15. Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH).

    PubMed

    Vági, Pál; Knapp, Dániel G; Kósa, Annamária; Seress, Diána; Horváth, Áron N; Kovács, Gábor M

    2014-05-01

    In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant-fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period. PMID:24221902

  16. Protecting Quantum Dot Fluorescence from Quenching to Achieve a Reliable Automated Multiplex Fluorescence In Situ Hybridization Assay.

    PubMed

    Zhang, Wenjun; Hubbard, Antony; Pang, Lizhen; Parkinson, Leslie Baca; Brunhoeber, Patrick; Wang, Yixin; Tang, Lei

    2015-09-01

    Quantum dots (QD) are novel inorganic fluorochromes that are ultra-bright, photo-stable, and available in multiple, highly-resolvable colors. QDs represent an ideal detection material for in situ hybridization (ISH) because they may provide unprecedented resolution and strong signal intensities that are not attainable with traditional fluorophores. Unfortunately, lack of reliability has been an impediment to widespread adoption of QD-based fluorescence in situ hybridization (QD FISH) technology. By optimizing QD-to-target accessibility, we have developed a QD FISH staining procedure that dramatically improves the reliability of an automated ERG/PTEN QD FISH assay (91% 1st pass rate). Here, we report improvements to the assay that protects QD fluorescence from quenching due to trace amounts of heavy metals and minimizes QD background signals. When using this method, highly-consistent staining was observed with the ERG/PTEN QD FISH assay in prostate tissue. Successful staining of several other clinically-relevant genetic markers was also possible. We further demonstrated improved reliability for determining HER2 gene status in breast cancer, identifying anaplastic lymphoma kinase (ALK) gene break-apart in non-small cell lung cancer, and detecting human papillomavirus 16 (HPV16) in cervical intraepithelial neoplasia. The enhanced QD FISH assay allows for examining complicated genetic aberrances without use of enzymatic amplification. Our optimized methods now demonstrate reliability sufficient for QD FISH technology to be a diagnostic tool in a clinical setting. PMID:26485928

  17. Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy.

    PubMed

    Markaki, Yolanda; Smeets, Daniel; Cremer, Marion; Schermelleh, Lothar

    2013-01-01

    Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging. Three-dimensional structured illumination microscopy (3D-SIM) represents one of these technologies. Notably, 3D-SIM renders an eightfold improved volumetric resolution over conventional imaging, and allows the simultaneous visualization of differently labeled target structures. These features make this approach highly attractive for the analysis of spatial relations and substructures of nuclear targets that escape detection by conventional light microscopy. Here, we focus on the application of 3D-SIM for the visualization of subnuclear 3D-FISH preparations. In comparison with conventional fluorescence microscopy, the quality of 3D-SIM data is dependent to a much greater extent on the optimal sample preparation, labeling and acquisition conditions. We describe typical problems encountered with super-resolution imaging of in situ hybridizations in mammalian tissue culture cells and provide optimized DNA-/(RNA)-FISH protocols including combinations with immunofluorescence staining (Immuno-FISH) and DNA replication labeling using click chemistry. PMID:23086869

  18. Detection of a complex translocation using fluorescent in situ hybridization (FISH)

    SciTech Connect

    Rosen, B.A.; Abuelo, D.N.; Mark, H.F.

    1994-09-01

    The use of fluorescent in situ hybridization (FISH) allowed the detection of a complex 3-way translocation in a patient with multiple congenital malformations and mental retardation. The patient was a 10-year-old girl with mental retardation, seizures, repaired cleft palate, esotropia, epicanthal folds, broad nasal bridge, upward slanting palpebral fissures, single transverse palmar crease, brachydactyly, hypoplastic nails, ectrodactyly between the third and fourth right toes, and hypoplasia of the left third toe. Chromosome analysis performed at birth was reported as normal. We performed high resolution banding analysis which revealed an apparently balanced translocation between chromosomes 2 and 9. However, because of her multiple abnormalities, further studies were ordered. Fluorescent in situ hybridization (FISH) using chromosome painting probes revealed a karyotype of 46,XX,t(2;8;9) (2pter{yields}q31::8q21.2{yields}8qter; 8pter{yields}q21.2::2q31{yields}q34::9q34{yields}qter; 9pter{yields}q34::2q34{yields}qter). The 3-way translocation appears to be de novo, as neither parent is a translocation carrier. This case illustrates the importance of using FISH to further investigate cases of apparently balanced translocations in the presence of phenotypic abnormalities and/or mental retardation.

  19. Fluorescent in situ hybridization analyses of human oocytes in trisomy 18 and 21

    SciTech Connect

    Cheng, E.Y.; Chen, Y.J.; Gartler, S.M.

    1994-09-01

    The commonly accepted view of synapsis is that only 2 homologues can synapse at any one site and that this restriction applies to polyploids as well. However, triple synapsis has been observed is some triploid plants and in triploid chicken. In humans, triple synapsis of the long arm of chromosome 21 was detected in sperm of a trisomic 21 individual. More recently, studies of oocytes from trisomic 21 and 18 fetuses also indicated extensive triple synapsis along the entire length of the chromosomes. To further investigate this question, we undertook an evaluation of trivalent synapsis in fetal oocytes from 2 trisomic 21 and 2 trisomic 18 fetuses using fluorescent in situ hybridization (FISH) with whole chromosome probes. Oocytes were hybridized with whole chromosome probes obtained from ONCOR, Inc. after fixation with methanol and acetic acid. Slides were scored for the distribution of prophase stages, hybridization efficiency, and hybridization characteristics of chromosomes 18 and 21 in the trisomic 18 and 21 fetuses respectively. Fifty-eight per cent (379/650) of pachytenes analyzed for chromosome 18 contained a conspicous trivalent and 319 (48%) of these nuclei contained a single, thick, continuous fluorescent signal consistent with complete triple synapsis along the entire length of all 3 chromosomes. Sixteen per cent (104/650) of pachytene contained 2 signals consistent with a bivalent and a univalent, and 9 cells contained 3 thin signals consistent with asynapsis of all 3 chromosomes. The remaining 158 pachytenes had unusual pairing configurations that we could not classify, but they most likely represent trivalents with partial pairing between different homologues. In the 2 trisomic 21 fetuses, the majority (143/232) of pachytenes also contained one signal while only 52 cells contained a bivalent and univalent. Five cells contained 3 separate signals. These results confirm the existence of triple synapsis in human meiosis.

  20. Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: A

    E-print Network

    Wu, Yih-Min

    Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid in model organisms. Previously, we developed a robust protocol for whole- mount RNA CISH in the pea aphid of germline specification and embryonic segmentation in the pea aphid. We anticipate that the RNA

  1. Chromosome-specific DNA repeats: rapid identification in silico and validation using fluorescence in situ hybridization.

    PubMed

    Hsu, Joanne H; Zeng, Hui; Lemke, Kalistyn H; Polyzos, Aris A; Weier, Jingly F; Wang, Mei; Lawin-O'Brien, Anna R; Weier, Heinz-Ulrich G; O'Brien, Benjamin

    2012-01-01

    Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH) is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as "database mining". Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols. PMID:23344021

  2. Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

    PubMed Central

    Hsu, Joanne H.; Zeng, Hui; Lemke, Kalistyn H.; Polyzos, Aris A.; Weier, Jingly F.; Wang, Mei; Lawin-O’Brien, Anna R.; Weier, Heinz-Ulrich G.; O’Brien, Benjamin

    2013-01-01

    Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH) is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols. PMID:23344021

  3. Chromogenic in situ hybridization is a reliable alternative to fluorescence in situ hybridization for diagnostic testing of 1p and 19q loss in paraffin-embedded gliomas.

    PubMed

    Lass, Ulrike; Hartmann, Christian; Capper, David; Herold-Mende, Christel; von Deimling, Andreas; Meiboom, Maren; Mueller, Wolf

    2013-05-01

    Recent studies imply the importance of rapid and reliable diagnostic assessment of 1p/19q status in oligodendroglial tumors. To date, fluorescence in situ hybridization (FISH) is the most commonly applied technique. FISH, however, has several technical shortcomings that are suboptimal for diagnostic applications: results must be viewed in a fluorescence microscope, results are usually evaluated by a single investigator only, and signal fading excludes physical archiving. Also, in gliomas, the distinction of diffusely infiltrating tumor cells from reactively altered normal tissue may be challenging in fluorescence microscopy. Dual-color chromogenic in situ hybridization (CISH) has started to replace FISH in some diagnostic tests performed in pathology. Here, we present the first single institute experience with a side-by-side analysis of 1p/19q FISH and CISH in a series of 42 consecutive gliomas. FISH and CISH produced identical results for 1p and 19q in 93% of cases (n?=?39/42). Discrepant results were reevaluated by repeated FISH and a polymerase chain reaction (PCR)-based microsatellite marker analysis for loss of heterozygosity. Reevaluation confirmed CISH data in all three cases. We conclude that CISH is a reliable alternative in 1p/19q testing in paraffin-embedded tissues likely to be more sensitive to detect 1p/19q status than FISH analysis. PMID:23107103

  4. Identification of supernumerary ring chromosome 1 mosaicism using fluorescence in situ hybridization

    SciTech Connect

    Chen, H.; Tuck-Muller, C.M.; Wertelecki, W.

    1995-03-27

    We report on a 15-year-old black boy with severe mental retardation, multiple congenital anomalies, and a supernumerary ring chromosome mosaicism. Fluorescence in situ hybridization with a chromosome 1 painting probe (pBS1) identified the ring as derived from chromosome 1. The karyotype was 46,XY/47,XY,+r(1)(p13q23). A review showed 8 reports of ring chromosome 1. In 5 cases, the patients had a non-supernumerary ring chromosome 1 resulting in partial monosomies of the short and/or long arm of chromosome 1. In 3 cases, the presence of a supernumerary ring resulted in partial trisomy of different segments of chromosome 1. In one of these cases of the supernumerary ring was composed primarily of the centromere and the heterochromatic region of chromosome 1, resulting in normal phenotype. Our patient represents the third report of a supernumerary ring chromosome 1 resulting in abnormal phenotype. 28 refs., 5 figs., 1 tab.

  5. Chromosome translocations measured by fluorescence in-situ hybridization: A promising biomarker

    SciTech Connect

    Lucas, J.N.; Straume, T.

    1995-10-01

    A biomarker for exposure and risk assessment would be most useful if it employs an endpoint that is highly quantitative, is stable with time, and is relevant to human risk. Recent advances in chromosome staining using fluorescence in situ hybridization (FISH) facilitate fast and reliable measurement of reciprocal translocations, a kind of DNA damage linked to both prior exposure and risk. In contrast to other biomarkers available, the frequency of reciprocal translocations in individuals exposed to whole-body radiation is stable with time post exposure, has a rather small inter-individual variability, and can be measured accurately at the low levels. Here, the authors discuss results from their studies demonstrating that chromosome painting can be used to reconstruct radiation dose for workers exposed within the dose limits, for individuals exposed a long time ago, and even for those who have been diagnosed with leukemia but not yet undergone therapy.

  6. The design of a microscopic system for typical fluorescent in-situ hybridization applications

    NASA Astrophysics Data System (ADS)

    Yi, Dingrong; Xie, Shaochuan

    2013-12-01

    Fluorescence in situ hybridization (FISH) is a modern molecular biology technique used for the detection of genetic abnormalities in terms of the number and structure of chromosomes and genes. The FISH technique is typically employed for prenatal diagnosis of congenital dementia in the Obstetrics and Genecology department. It is also routinely used to pick up qualifying breast cancer patients that are known to be highly curable by the prescription of Her2 targeted therapy. During the microscopic observation phase, the technician needs to count typically green probe dots and red probe dots contained in a single nucleus and calculate their ratio. This procedure need to be done to over hundreds of nuclei. Successful implementation of FISH tests critically depends on a suitable fluorescent microscope which is primarily imported from overseas due to the complexity of such a system beyond the maturity of the domestic optoelectrical industry. In this paper, the typical requirements of a fluorescent microscope that is suitable for FISH applications are first reviewed. The focus of this paper is on the system design and computational methods of an automatic florescent microscopy with high magnification APO objectives, a fast spinning automatic filter wheel, an automatic shutter, a cooled CCD camera used as a photo-detector, and a software platform for image acquisition, registration, pseudo-color generation, multi-channel fusing and multi-focus fusion. Preliminary results from FISH experiments indicate that this system satisfies routine FISH microscopic observation tasks.

  7. Fluorescence in situ hybridization for the identification of Treponema pallidum in tissue sections.

    PubMed

    Petrich, Annett; Rojas, Pablo; Schulze, Julia; Loddenkemper, Christoph; Giacani, Lorenzo; Schneider, Thomas; Hertel, Moritz; Kikhney, Judith; Moter, Annette

    2015-10-01

    Syphilis is often called the great imitator because of its frequent atypical clinical manifestations that make the disease difficult to recognize. Because Treponema pallidum subsp. pallidum, the infectious agent of syphilis, is yet uncultivated in vitro, diagnosis is usually made using serology; however, in cases where serology is inconclusive or in patients with immunosuppression where these tests may be difficult to interpret, the availability of a molecular tool for direct diagnosis may be of pivotal importance. Here we present a fluorescence in situ hybridization (FISH) assay that simultaneously identifies and analyzes spatial distribution of T. pallidum in histological tissue sections. For this assay the species-specific FISH probe TPALL targeting the 16S rRNA of T. pallidum was designed in silico and evaluated using T. pallidum infected rabbit testicular tissue and a panel of non-syphilis spirochetes as positive and negative controls, respectively, before application to samples from four syphilis-patients. In a HIV positive patient, FISH showed the presence of T. pallidum in inguinal lymph node tissue. In a patient not suspected to suffer from syphilis but underwent surgery for phimosis, numerous T. pallidum cells were found in preputial tissue. In two cases with oral involvement, FISH was able to differentiate T. pallidum from oral treponemes and showed infection of the oral mucosa and tonsils, respectively. The TPALL FISH probe is now readily available for in situ identification of T. pallidum in selected clinical samples as well as T. pallidum research applications and animal models. PMID:26365167

  8. A 30-Mb metric fluorescence in situ hybridization map of human chromosome 19q

    SciTech Connect

    Gordon, L.A.; Bergmann, A.; Christensen, M.; Danganan, L.

    1995-11-20

    A high-resolution metric physical map of chromosome 19q has been constructed by fluorescence in situ hybridization. The map locates 136 cosmid reference points that span 30 Mb. The reference points are sequentially ordered from centromere to telomere, and the distance between neighboring cosmids is known from 240 partially overlapping, redundant estimates of genomic distances in kilobases separating pairs of cosmids. The average spacing between cosmid reference points is 220 kb, with over 75% of intervals less than 300 kb. Eighty-four genes and polymorphic markers have been assigned to mapped cosmids. The information on order and genomic distances separating pairs of cosmids, both key elements for building physical maps, has furthered the construction and integration of the genetic and physical maps of chromosome 19. 24 refs., 3 figs.

  9. Partial trisomy 13q identified by sequential fluorescence in situ hybridization

    SciTech Connect

    Gopal Rao, V.V.N.; Carpenter, N.J.; Gucsavas, M.

    1995-07-31

    We report on a 19-month-old boy with partial trisomy 13q resulting from a probable balanced translocation involving chromosomes 1 and 13. The infant presented with omphalocele, malrotation, microcephaly with overriding skull bones, micrognathia, apparently low-set ears, rocker-bottom feet, and congenital heart disease, findings suggestive of trisomy 13. Karyotypic studies from peripheral blood lymphocytes documented an unbalanced karyotype 46,XY,-1,+der. The mother`s chromosomes were normal, and the father was not available. Conventional cytogenetic techniques were unable to identify the extra material on the terminal 1q. Using fluorescence in situ hybridization (FISH) on the GTL-banded metaphases, the extra material on 1q was identified as the terminal long arm of 13, thus resulting in partial trisomy 13 (q32-qter). 8 refs., 2 figs., 1 tab.

  10. Development of a fluorescent in situ hybridization (FISH) technique for visualizing CGMMV in plant tissues.

    PubMed

    Shargil, D; Zemach, H; Belausov, E; Lachman, O; Kamenetsky, R; Dombrovsky, A

    2015-10-01

    Cucumber green mottle mosaic virus (CGMMV), which belongs to the genus Tobamovirus, is a major pathogen of cucurbit crops grown indoors and in open fields. Currently, immunology (e.g., ELISA) and molecular amplification techniques (e.g., RT-PCR) are employed extensively for virus detection in plant tissues and commercial seed lots diagnostics. In this study, a fluorescent in situ hybridization (FISH) technique, using oligonucleotides whose 5'-terminals were labeled with red cyanine 3 (Cy3) or green fluorescein isothiocyanate (FITC), was developed for the visualization of the pathogen in situ. This simple and reliable method allows detection and localization of CGMMV in the vegetative and reproductive tissues of cucumber and melon. When this technique was applied in male flowers, anther tissues were found to be infected; whereas the pollen grains were found to be virus-free. These results have meaningful epidemiological implications for the management of CGMMV, particularly with regard to virus transfer via seed and the role of insects as CGMMV vectors. PMID:26231788

  11. A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis

    SciTech Connect

    Stokke, T.; Collins, C.; Kuo, W.L.

    1995-03-01

    The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously. 14 refs., 1 fig., 2 tabs.

  12. Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH)

    PubMed Central

    Lecrenier, M. C.; Ledoux, Q.; Berben, G.; Fumière, O.; Saegerman, C.; Baeten, V.; Veys, P.

    2014-01-01

    Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as “mad cow disease”), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application. PMID:25034259

  13. Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).

    PubMed

    Lecrenier, M C; Ledoux, Q; Berben, G; Fumière, O; Saegerman, C; Baeten, V; Veys, P

    2014-01-01

    Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as "mad cow disease"), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application. PMID:25034259

  14. RAPID IDENTIFICATION OF CANDIDA ALBICANS DIRECTLY FROM YEAST POSITIVE BLOOD CULTURE BOTTLES BY FLUORESCENCE IN SITU HYBRIDIZATION USING PNA PROBES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for identification of Candida albicans directly from yeast-positive blood culture bottles is described. The test (C. albicans PNA FISH) is based on a fluorescein-labeled PNA probe targeting C. albicans 26...

  15. Generation of an integrated karyotype of the honey bee (Apis mellifera L.) by banding pattern and fluorescent in situ hybridization 

    E-print Network

    Aquino Perez, Gildardo

    2009-05-15

    . The primary analysis of the karyotype and ideogram construction was based on banding and Fluorescence In Situ Hybridization (FISH) for rDNA detection. FISH confirmed two locations for the NOR on telomeric regions of chromosomes 6 and 12 plus an additional less...

  16. X chromosome aneuploidy in infertile women: Analysis by interphase fluorescent in situ hybridization

    SciTech Connect

    Morris, M.A.; Moix, I.; Mermillod, B.

    1994-09-01

    Up to 1 in 3 couples have a problem of infertility at some time in their lives. Sex chromosome anomalies are found in 5-10% of couples, with mosaic aneuploidy being a common finding in primary infertility. Recurrent spontaneous abortion (RSA), in contrast, is frequently associated with autosomal structural anomalies. We hypothesized that low-level mosaic X chromosome aneuploidy was associated with primary infertility but not with RSA. Three groups were studied: women from couples with primary infertillity (n=26); women with three or more spontaneous abortions (n=22); and age-matched normally fertile women (at least two pregnancies; n=28). Interphase fluorescent in situ hybridization (FISH) was used to determine X chromosome ploidy in 100 nuclei per patient, using a contig of three cosmids from MAO locus (kindly donated by W. Berger, Nijmegen). A control probe (chr. 15 centromere) was simultaneously hybridized, and only nuclei containing two control signals were scored for the X chromosome. The mean numbers of nuclei with two X chromosome signals were the same in all groups (Welch equality of means test: p>0.97). However, there is a significant difference between the variances of the primary infertile and RSA groups (Levene`s test: p=0.025 after Bonferrone correction for multiple testing). This provides preliminary support for the hypothesis of an association between primary infertility and low-level mosaic X chromosome aneuploidy.

  17. Fluorescent in situ hybridization (FISH) assessment of chromosome copy number in sperm

    SciTech Connect

    Sheu, M.; Sigman, M.; Mark, H.F.L.

    1994-09-01

    Approximately 15% of all recognized pregnancies end in spontaneous abortions. The overall frequency of chromosome abnormalities in spontaneous abortions is approximately 50%. Thus aneuploidy is a significant cause of fetal wastage. In addition, structural and numerical abnormalities of chromosomes can also lead to birth defects, developmental delay, mental retardation and infertility. Conventional cytogenetic analysis via GTG- and other banding techniques is a powerful tool in the elucidation of the nature of chromosomal abnormalities. Fluorescent in situ hybridization (FISH) enables detection of numerical chromosomal abnormalities, especially trisomies, in intact cells. Using FISH and commercially available biotin-labeled probes, we have initiated a prospective study to assess specific chromosome copy number of preparations of unstained smears from men referred for a male infertility evaluation as well as smears from normal control males chosen randomly from the sample of sperm donors. A total of approximately 19,000 sperm nuclei have been examined thus far. Of those suitable for analysis, 7382 (38.75%) were normal possessing one copy of chromosome 8, 155 (0.81%) were disomic, and 15 (0.079%) had more than two copies of chromosome 8. Comparisons with data available in the literature will be discussed. Work is ongoing to increase the efficiency of hybridization using both reported and previously untried pretreatment and fixation protocols. We have also initiated studies using multicolor FISH with various chromosome enumeration probes. The assay described here is a potentially powerful tool for detecting rare events such as spontaneous germ cell aneuploidy, aneuploidy detected in semen from men with carcinoma in situ of the testis and aneuploidy induced by potential environmental genotoxicants. It can also be utilized for segregation analysis and for correlating chromosome copy number with germ cell morphology.

  18. Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis.

    PubMed

    Milinovich, Gabriel J; Trott, Darren J; Burrell, Paul C; Croser, Emma L; Al Jassim, Rafat A M; Morton, John M; van Eps, Andrew W; Pollitt, Christopher C

    2007-08-01

    Carbohydrate-induced laminitis in horses is characterized by marked changes in the composition of the hindgut microbiota, from a predominantly Gram-negative population to one dominated by Gram-positive bacteria. The objective of this study was to monitor changes in the relative abundance of selected hindgut bacteria that have previously been implicated in the pathophysiology of equine laminitis using fluorescence in situ hybridization (FISH). Caecal cannulae were surgically implanted in five Standardbred horses and laminitis induced by oral administration of a bolus dose of oligofructose. Caecal fluid and faecal specimens were collected over a 48 h period at 2 to 4 h intervals post-oligofructose administration and subjected to FISH using probes specific for nine bacterial groups to determine changes in their relative abundance compared with total bacteria hybridizing to the generic EUBMIX probe. Additionally, hoof biopsies were taken over the course of the experiment at 6 h intervals and evaluated for histopathological changes consistent with laminitis, allowing changes in hindgut microbiota to be correlated with the onset of lesions in the foot. Of the microorganisms specifically targeted, streptococci of the Streptococcus bovis/equinus complex were the only bacteria that consistently proliferated in both caecal fluid and faeces immediately before the onset of histological signs of laminitis. Furthermore, lactobacilli, Enterobacteriaceae, Allisonella histaminiformans, enterococci, Bacteroides fragilis, Mitsuokella jalaludinii and Clostridium difficile did not establish significant populations in the hindgut before the onset of equine laminitis. PMID:17635552

  19. Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting

    E-print Network

    Chen, Wilfred

    METHODS Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluores- cence in situ to the respective targets, with minimal non- specific binding. The recombinant strain was inoculated into wheat

  20. Identification of pathogens in mastitis milk samples with fluorescent in situ hybridization.

    PubMed

    Gey, Annerose; Werckenthin, Christiane; Poppert, Sven; Straubinger, Reinhard K

    2013-05-01

    Traditionally, the bacteriological examination of mastitis milk samples is performed by culture followed by biochemical tests on the cultured bacteria to allow identification of the causative pathogen. Depending on the species involved, this classic identification is time-consuming compared to other techniques such as fluorescent in situ hybridization (FISH), a culture-independent method that utilizes oligonucleotides (labeled with a fluorophore) that are specific to a string of target DNA/RNA. In the current study, the applicability of FISH was evaluated for the detection of mastitis pathogens directly in milk samples. To remove interfering lipids and proteins from mastitis milk samples prior to FISH, a previously published enzymatic treatment with savinase was evaluated. FISH was performed using oligonucleotides specific for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Trueperella (Arcanobacterium) pyogenes. The enzymatic pretreatment and the sensitivity of FISH were evaluated using spiked whole milk samples and mastitis milk samples with bacterial loads of less than 10(3) up to 10(8) colony-forming units (CFU)/ml. Bacteria were reliably detected in milk samples with bacterial numbers of 10(6) CFU/ml or higher. However, bacteria present in numbers below 10(6) CFU/ml were not detectable in all cases. The ability of FISH to identify mastitis-causing pathogens directly in milk samples, and therefore earlier than classical culture methods, can supplement the classic diagnostic procedures for mastitis milk samples. PMID:23632662

  1. Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

    PubMed Central

    Shah, Jyotsna; Mark, Olivia; Weltman, Helena; Barcelo, Nicolas; Lo, Wai; Wronska, Danuta; Kakkilaya, Srinivas; Rao, Aravinda; Bhat, Shalia T.; Sinha, Ruchi; Omar, Sabah; Moro, Manuel; Gilman, Robert H.; Harris, Nick

    2015-01-01

    Malaria is a responsible for approximately 600 thousand deaths worldwide every year. Appropriate and timely treatment of malaria can prevent deaths but is dependent on accurate and rapid diagnosis of the infection. Currently, microscopic examination of the Giemsa stained blood smears is the method of choice for diagnosing malaria. Although it has limited sensitivity and specificity in field conditions, it still remains the gold standard for the diagnosis of malaria. Here, we report the development of a fluorescence in situ hybridization (FISH) based method for detecting malaria infection in blood smears and describe the use of an LED light source that makes the method suitable for use in resource-limited malaria endemic countries. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probe that detects all five species of Plasmodium known to cause the disease in humans. The P. falciparum (PF) FISH assay and P. vivax (PV) FISH assay detect and differentiate between P. falciparum and P. vivax respectively from other Plasmodium species. The FISH assays are more sensitive than Giemsa. The sensitivities of P-Genus, PF and PV FISH assays were found to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of Plasmodium, P. falciparum and P. vivax by Giemsa staining respectively. PMID:26333092

  2. Investigation of chromosomal aberrations in hepatocellular carcinoma by fluorescence in situ hybridization.

    PubMed

    Huang, S F; Hsu, H C; Fletcher, J A

    1999-05-01

    Molecular cytogenetic approaches have been applied only rarely in the characterization of hepatocellular carcinoma (HCC). The aim in this study was to evaluate aberrations, particularly deletions, of specific chromosomal regions in HCC. Dual-color fluorescence in situ hybridization (FISH) was performed on intact nuclei from touch preparations of 17 HCCs and 1 hepatic adenoma. Each touch preparation was hybridized with a digoxigenin-labeled centromere probe and a biotin-labeled unique sequence probe from the same chromosome. This approach permitted the simultaneous evaluation of ploidy changes and chromosome arm deletions. Eight noncentromeric chromosome regions, 3p14, 4q21, 6q14, 6q21, 8p12, 8p22, 9p21, and 9p24 were selected for study on the basis of their having been implicated as tumor suppressor regions in HCC or other common types of carcinoma. Together with the 5 corresponding centromeric probes on chromosomes 3, 4, 6, 8, and 9, a total of 13 chromosome loci were evaluated. All cases of hepatocellular carcinoma showed at least one deletion or aneuploidy. The hepatic adenoma was all diploid. Chromosome 4q21 showed the highest rate of deletion (76.5%) and aneusomy (88%). The second and the third were chromosome 8p22 and 6q14, which showed 59% and 47% of deletion, respectively. A 4q21 deletion is also the most frequent single chromosome aberration. Prominent tumor heterogeneity and variable deletion patterns were noted. Interphase FISH was an efficient means for evaluating numerical and structural chromosome aberrations in HCCs. Most HCCs contained deletions of known tumor suppressor regions (4q and 8p), and a novel deletion hotspot was demonstrated on chromosome band 6q14. PMID:10326586

  3. Molecular characterization of the breakpoints in rob(13q14q) by fluorescence in situ hybridization

    SciTech Connect

    Han, J.Y.; Shaffer, L.G.; Choo, K.H.A.

    1994-09-01

    Robertsonian translocations are the most common rearrangements in humans with rob(13q14q) contributing to the majority of all ascertained rearrangements. Studying the sequences around the breakpoint regions of Robertsonian translocations may enable us to understand the underlying mechanisms of translocation formation and the molecular organization of the centromeric and pericentromeric regions of the acrocentric chromosomes. We have characterized 17 rob(13q14q), including 6 de novo rearrangements, 5 maternally and 3 paternally inherited rearrangements, and 3 of undetermined origin, by dual color fluorescence in situ hybridization (FISH) and 6 molecular probes which are specific for the repetitive sequences in the centromeric and short arm regions of acrocentric chromosomes. The centromeric alpha satellite DNA probes D21Z1/D13Z1 and D14Z1/D22Z1 showed all rob(13q14q) chromosomes to be dicentric. The rDNA probe did not hybridize to the 17 translocations studied. The pTRS-47 satellite III DNA probe specific for chromosomes 14 and 22 indicated the retention of pTRS-47 sequences around the breakpoints in all cases studied. However, pTRS-63 satellite III probe specific for chromosome 14 did not show any signals on the translocation chromosomes. In 16 of 17 translocations, strong hybridization signals were detected with pTRI-6 satellite I DNA probe specific for chromosome 13. All parental chromosomes 13 and 14 for 6 de novo rob(13q14q), including one whose pTRI-6 sequence was lost, showed strong hybridization signals with pTRS-47 and pTRS-63, and pTRI-6 probes, respectively. These results demonstrate that the breakpoints fall between the pTRS-47 and pTRS-63 sequences in the p11 region of chromosome 14 and between the pTRI-6 and rDNA sequences within the p11 region of chromosome 13 further narrowing the region containing the rob(13q14q) breakpoints.

  4. Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

    PubMed

    Kiyose, Shinichiro; Igarashi, Hisaki; Nagura, Kiyoko; Kamo, Takaharu; Kawane, Kazunori; Mori, Hiroki; Ozawa, Takachika; Maeda, Matsuyoshi; Konno, Keisuke; Hoshino, Hideaki; Konno, Hiroyuki; Ogura, Hiroyuki; Shinmura, Kazuya; Hattori, Naohiko; Sugimura, Haruhiko

    2012-11-01

    The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples. PMID:23121603

  5. Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine

    PubMed Central

    2014-01-01

    Extensive studies of the genetic aberrations related to human diseases conducted over the last two decades have identified recurrent genomic abnormalities as potential driving factors underlying a variety of cancers. Over the time, a series of cutting-edge high-throughput genetic tests, such as microarrays and next-generation sequencing, have been developed and incorporated into routine clinical practice. Although it is a classical low-throughput cytogenetic test, fluorescence in situ hybridization (FISH) does not show signs of fading; on the contrary, it plays an increasingly important role in detecting specific biomarkers in solid and hematologic neoplasms and has therefore become an indispensable part of the rapidly developing field of personalized medicine. In this article, we have summarized the recent advances in FISH application for both de novo discovery and routine detection of chromosomal rearrangements, amplifications, and deletions that are associated with the pathogenesis of various hematopoietic and non-hematopoietic malignancies. In addition, we have reviewed the recent developments in FISH methodology as well. PMID:24499728

  6. Potential clinical impact of three-dimensional visualization for fluorescent in situ hybridization image analysis

    NASA Astrophysics Data System (ADS)

    Li, Zheng; Li, Shibo; Bin, Zheng; Zhang, Roy; Li, Yuhua; Tian, Huimin; Chen, Wei; Liu, Hong

    2012-05-01

    Chromosomal translocation is strong indication of cancers. Fluorescent in situ hybridization (FISH) can effectively detect this translocation and achieve high accuracy in disease diagnosis and prognosis assessment. For this purpose, whole chromosome paint probes are utilized to image the configuration of DNA fragments. Although two-dimensional (2-D) microscopic images are typically used in FISH signal analysis, we present a case where the translocation occurs in the depth direction where two probed FISH signals are overlapped in the projected image plane. Thus, the translocation cannot be identified. However, when imaging the whole specimen with a confocal microscope at 27 focal planes with 0.5-?m step interval, the translocation can be clearly identified due to the free rotation capability by the three-dimensional (3-D) visualization. Such a translocation detection error of using 2-D images might be critical in detecting and diagnosing early or subtle disease cases where detecting a small number of abnormal cells can make diagnostic difference. Hence, the underlying implication of this report suggests that utilizing 3-D visualization may improve the overall accuracy of FISH analysis for some clinical cases. However, the clinical efficiency and cost of using 3-D versus 2-D imaging methods are also to be assessed carefully.

  7. [An improved protocol of preparing bone marrow cells for fluorescence in situ hybridization].

    PubMed

    Hu, Lin-Ping; Ge, Jing; Zhang, Li-Yan; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao; Zhang, Lei

    2012-04-01

    This study was aimed to establish a smear protocol for preparing bone marrow cells and investigate its effect on fluorescence in situ hybridization (FISH) signal. Probe DNA (C-myc, MDM2, STK6) was labeled with Spectrum Green, PromoFluor-555 and PromoFluor-415 by nick translation. Five bone marrow samples were tested by two methods separately. Traditional method: after removing the erythrocytes by hypoosmotic solution, the bone marrow cells were fixed in methanol/acetic acid (3:1). Improved method: erythrocytes were removed using density gradient centrifugation and fixed in methanol. The samples were then fixed again in 2 formaldehyde for 5 min. The FISH signal was assessed by comparing the relative signal intensity of each fluorophore with the autofluorescence background. The results indicated that improved method greatly increased the ratio of fluorescence signal intensity in the Spectrum Green, PromoFluor-555 and PromoFluor-415 channel (traditional method: 4.3 ± 0.19, 3.52 ± 0.04, 3.07 ± 0.08; improved method: 9.89 ± 0.41, 7.55 ± 0.5, 5.67 ± 0.18, n = 5, P < 0.01) respectively. The signal intensity increased 2.32, 2.14 and 1.85-fold in the Spectrum Green, PromoFluor-555 and PromoFluor-415 channel respectively. In addition, the improved method decreased the split signals [traditional method: (15.8 ± 1.74), (20.42 ± 2.88), (23.2 ± 3.02); improved method: (8.6 ± 1.2), (12.28 ± 1.33), (12.6 ± 2.56), n = 5, P < 0.05]. It is concluded that the improved optimal procedure which facilitates FISH intensity on bone marrow cells is developed, showing potential for wide application in the diagnosis of hematologic diseases. PMID:22541126

  8. Estimate of true incomplete exchanges using fluorescence in situ hybridization with telomere probes

    NASA Technical Reports Server (NTRS)

    Wu, H.; George, K.; Yang, T. C.

    1998-01-01

    PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

  9. Interphase fluorescence in situ hybridization signal detection by computing intensity variance along the optical axis

    NASA Astrophysics Data System (ADS)

    Li, Zheng; Zheng, Bin; Ren, Liqiang; Liu, Hong

    2014-02-01

    Fluorescence in situ Hybridization technology is a commonly used tool to detect chromosome aberrations, which are often pathologically significant. Since manual FISH analysis is a tedious and time-consuming procedure, reliable and robust automated image acquisition and analysis are in demand. Under high magnification objective lenses such as 60x and 100x, the depth of field will often be too small and the FISH probes may not always lie in the same focal plane. A statistical variance based automated FISH analysis method is developed in order to address this problem. On a stack of slices at consecutive image planes with a step size d, the statistical variance alone the z-axis is calculated to form a 2-D matrix. Since pixels shift dramatically to high intensity at FISH probe location, the probes will manifest high peak values in the matrix. A computer-aided detection scheme based on top-hat transform is applied to the matrix to detect FISH probe signals. This study demonstrates a simple and robust method for FISH probe detection as well as a way of 2- D representation of 3-D data.

  10. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method.

    PubMed

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina; Cerca, Nuno

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6-99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  11. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  12. Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.

    PubMed

    Williamson, Iain; Berlivet, Soizik; Eskeland, Ragnhild; Boyle, Shelagh; Illingworth, Robert S; Paquette, Denis; Dostie, Josée; Bickmore, Wendy A

    2014-12-15

    Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods. PMID:25512564

  13. Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

    PubMed Central

    Williamson, Iain; Berlivet, Soizik; Eskeland, Ragnhild; Boyle, Shelagh; Illingworth, Robert S.; Paquette, Denis

    2014-01-01

    Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods. PMID:25512564

  14. Microfluidic fluorescence in situ hybridization and flow cytometry (µFlowFISH)

    PubMed Central

    Liu, Peng; Meagher, Robert J.; Light, Yooli Kim; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P.; Hazen, Terry C.; Singh, Anup K.

    2011-01-01

    We describe an integrated microfluidic device (µFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(VI) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The µFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed; and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp. strain RCH2 that are involved in Cr(VI) reduction and immobilization. Combined labeling and detection efficiencies of 74–97% were observed in experiments with simple mixtures of cultured cells confirmed specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of µFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford Site. We were able to monitor the numbers of Pseudomonas sp. with only 100–200 cells loaded into the microchip. The µFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

  15. Human chromosome 19p: a fluorescence in situ hybridization map with genomic distance estimates for 79 intervals spanning 20 Mb.

    PubMed

    Brandriff, B F; Gordon, L A; Fertitta, A; Olsen, A S; Christensen, M; Ashworth, L K; Nelson, D O; Carrano, A V; Mohrenweiser, H W

    1994-10-01

    A physical map of human chromosome 19p has been constructed by fluorescence in situ hybridization of cosmids to metaphase chromosomes and sperm pronuclear interphases. The map spans approximately 20 Mb and was generated with 141 multiple, partially overlapping estimates of genomic distances for 79 intervals separating 80 sequentially ordered cosmid reference points. The average distance separating pairs of cosmids was 250 kb, with a range from 50 to 700 kb; 75% of the intervals were estimated to be less than or equal to 300 kb and only 8 intervals were between 500 and 700 kb. Cosmids positive for 33 genes or gene families and 5 polymorphic markers were included among the mapped elements. The fluorescence in situ hybridization map will be useful for furthering the integration of the physical and genetic maps of 19p and for placing newly identified markers within a few hundred kb of their neighbors. PMID:7851886

  16. Fifty probands with extra structurally abnormal chromosomes characterized by fluorescence in situ hybridization

    SciTech Connect

    Blennow, E.; Telenius, H.; Nordenskjoeld, M.

    1995-01-02

    Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 (inv dup(15)), including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality. 42 refs., 2 figs., 2 tabs.

  17. Fluorescence In Situ Hybridization Analysis of Atypical Melanocytic Proliferations and Melanoma in Young Patients

    PubMed Central

    DeMarchis, Emilia H; Swetter, Susan M; Jennings, Charay D; Kim, Jinah

    2014-01-01

    Morphologic heterogeneity among melanocytic proliferations is a common challenge in the diagnosis of melanoma. In particular, atypical melanocytic lesions in children, adolescents, and young adults may be difficult to classify because of significant morphologic overlap with melanoma. Recently a four-probe fluorescence in situ hybridization (FISH) protocol to detect chromosomal abnormalities in chromosomes 6 and 11 has shown promise for improving the classification of melanocytic lesions. We sought to determine the correlation between FISH results, morphology, and clinical outcomes in a series of challenging melanocytic proliferations in young patients. We retrospectively performed the standard four-probe FISH analysis on 21 melanocytic neoplasms from 21 patients younger than 25 years of age (range 5–25 years, mean 14.6 years) from Stanford University Medical Center who were prospectively followed for a median of 51 months (range 1–136 months). The study cohort included patients with 5 confirmed melanomas, 2 melanocytic tumors of uncertain malignant potential (MelTUMPs), 10 morphologically challenging atypical Spitz tumors (ASTs), and 4 typical Spitz nevi. FISH detected chromosomal aberrations in all five melanomas and in one MelTUMP, in which the patient developed subsequent lymph node and distant metastasis. All 10 ASTs, 4 Spitz nevi, and 1 of 2 MelTUMPs were negative for significant gains or losses in chromosomes 6 and 11q. Our findings demonstrated a strong correlation between positive FISH results and the histomorphologic impression of melanoma. This finding was also true for the MelTUMP with poor clinical outcome. Therefore FISH may serve as a helpful adjunct in the classification of controversial melanocytic tumors in young patients. PMID:24924836

  18. Use of fluorescence in situ hybridization to clarify a complex chromosomal rearrangement in a child with multiple congenital anomalies

    SciTech Connect

    Spikes, A.S.; Hegmann, K.; Smith, J.L.

    1995-05-22

    A child with multiple congenital anomalies was referred for cytogenetic evaluation. G-banded analysis showed a complex chromosome rearrangement involving 6 different chromosomes and 10 breakpoints. Fluorescence in situ hybridization (FISH) using whole chromosome painting probes and repetitive sequence probes was performed. In most cases the painting probes alone helped to clarify the G-banded results. However, in one instance, where the terminal band of the long arm of chromosome 1 was involved, the use of a telomeric probe was essential in defining the rearrangement. 13 refs., 3 figs.

  19. Breakpoints in Robertsonian translocations are localized to satellite III DNA by fluorescence in situ hybridization

    SciTech Connect

    Gravholt, C.H.; Friedrich, U.; Caprani, M.; Jorgensen, A.L. )

    1992-12-01

    The authors characterized 21 t(13;14) and 3 t(14;21) Robertsonian translocations for the presence of DNA derived from the short arms of the translocated acrocentric chromosomes and identified their centromeres. Nineteen of these 24 translocation carriers were unrelated. Using centromeric [alpha]-repeat DNA as chromosome-specific probe, they found by in situ hybridization that all 24 translocation chromosomes were dicentric. The chromatin between the two centromeres did not stain with silver, and no hybridization signal was detected with probes for rDNA or [beta]-satellite DNA that flank the distal and proximal ends of the rDNA region on the short arm of the acrocentrics. By contrast, all 24 translocation chromosomes gave a distinct hybridization signal when satellite III DNA was used as probe. This result strongly suggests that the chromosomal rearrangements leading to Robertsonian translocations occur preferentially in satellite III DNA. The authors hypothesize that guanine-rich satellite III repeats may promote chromosomal recombination by formation of tetraplex structures. The findings localize satellite III DNA to the short arm of the acrocentric chromosomes distal to centromeric [alpha]-repeat DNA and proximal to [beta]-satellite DNA. 32 refs., 4 figs., 2 tabs.

  20. [Interphasic in situ fluorescent hybridization (FISH) in 4 cases of myeloid neoplasias with chromosome 7 changes].

    PubMed

    Arranz, E; Renedo, M; Ramos, C; Martínez, B; Prieto, E; Benítez, J

    1994-12-01

    The use of FISH as a complement to the conventional cytogenetic studies is of great help in attaining a better characterisation of the chromosome anomalies present in haematological malignancies, such as chromosome 7 monosomy. A study was carried out in three cases of acute non-lymphoblastic leukaemia and a myelodysplastic syndrome with chromosome 7 involvement, as shown by conventional cytogenetic studies. The Cocktail probe for chromosome 7 (DZ1, DZ2) was used (Oncor) in performing in situ hybridation. A monosomic cell line for chromosome 7, undetected by conventional techniques, was disclosed with this procedure in two of the cases. In the remaining two patients the monosomy of chromosome 7 was confirmed, although at percentages different from those attained with the conventional methods. PMID:7855698

  1. A simple and rapid fluorescence in situ hybridization microwave protocol for reliable dicentric chromosome analysis.

    PubMed

    Cartwright, Ian M; Genet, Matthew D; Kato, Takamitsu A

    2013-03-01

    Fluorescence in situhybridization (FISH) is an extremely effective and sensitive approach to analyzing chromosome aberrations. Until recently, this procedure has taken multiple days to complete. The introduction of telomeric and centromeric peptide nucleic acid (PNA) probes has reduced the procedure's duration to several hours, but the protocols still call for a high temperature (80-90°C) step followed by 1-3 h of hybridization. The newest method to speed up the FISH protocol is the use of a microwave to shorten the heating element to less than a minute; however this protocol still calls for a 1-h hybridization period. We have utilized PNA centromere/telomere probes in conjunction with a microwave oven to show telomere and centromere staining in as little as 30 s. We have optimized the hybridization conditions to increase the sensitivity and effectiveness of the new protocol and can effectively stain chromosomes in 2 min and 30 s of incubation. We have found that our new approach to FISH produces extremely clear and distinct signals. Radiation-induced dicentric formation in mouse and human fibroblast cells was analyzed by two individual scorers and the observed dicentrics matched very well. PMID:23161278

  2. Improved signal recognition for interphase fluorescent in-situ hybridization using a non-ionic detergent (NP-40) pretreatment

    SciTech Connect

    Zhu, H.M.; Day-Salvatore, D.L.; Sciorra, L.J.

    1994-09-01

    We have reported that the non-ionic detergent ethylphenolpoly (ethyleneglycolether)x known as Noniet-P40 (Shell International Petroleum) can gently disrupt cell membranes, resulting in cells with varying degrees of free chromatin release. The extent of this phenomena is dependent upon the concentration of NP-40 and the detergent`s exposure time to the cells. Treated cells can range from halos of DNA around the cells to fully extended free chromatin configurations. We have demonstrated that these treated cells are excellent targets for many different fluorescently labelled probes used for in situ hybridization studies. Recently, we have compared NP-40 harvested lymphocytes with normally harvested cells to see if we could improve upon the number of cells showing discreet signals in interphase fluorescent in situ hybridization. Preliminary work has shown that using a trisomy 21 cell line, one can get a statistically significant improvement with NP-40 pretreatment cells over control levels, in the number of cells having three discreet signals in interphase {open_quotes}FISH{close_quotes}. Such a pretreatment is simple to perform and may be of value when the number of cells available for analysis is low, as in the search for fetal cells from maternal circulation.

  3. Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)

    SciTech Connect

    Park, June-Woo; Tompsett, Amber; Zhang, Xiaowei; Newsted, John L.; Jones, Paul D.; Au, Doris; Kong, Richard; Wu, Rudolf S.S.; Giesy, John P. Hecker, Markus

    2008-10-15

    The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 {mu}g/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells.

  4. QUANTITATIVE IMAGING AND STATISTICAL ANALYSIS OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) OF AUREOBASIDIUM PULLULANS. (R823845)

    EPA Science Inventory

    Abstract

    Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal o...

  5. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes.

    PubMed

    Hwang, Gyoyeon; Lee, Hansol; Lee, Jiyeon

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. PMID:26449454

  6. Detection of aneuploidy in sperm of an ataxia telangiectasia patient using three-chromosome fluorescence in situ hybridization

    SciTech Connect

    Lowe, X.R.; Baulch, J.E.; Arnheim, N.

    1994-09-01

    Ataxia telangiectasia (A-T) is an inherited, recessive, cancer-prone disorder. Fluorescence in situ hybridization (FISH) with DNA probes specific for three chromosomes was applied to sperm of an A-T patient to determine if there may be an increased germinal risk for aneuploidy. Air-dried sperm smears were treated with proteinase K and were decondensed with DTT and LIS. The slides were then hybridized with fluorescently labeled repetitive DNA probes specific for chromosomes X, Y and 8, and a total of 11,825 sperm cells were scored. The ratio of sperm bearing X-8 and Y-8 was 1:1, as predicted. The frequencies of hyperhaploidy were 3.9, 1.0, 17.6 and 7.8 per 10,000 cells for categories X-X-8, Y-Y-8, X-Y-8 and 8-8-(X or Y), respectively, In addition, the frequency of diploidy (X-Y-8-8) was 18.6 and auto-diploidies (X-X-8-8 and Y-Y-8-8) were 1.0 and 2.0, respectively. These frequencies were not significantly different when compared with levels in healthy men (p > 0.1). Our finding suggests that chromosome X, Y and 8 aneuploidies are not elevated in the sperm of A-T patients, but studies with additional patients and chromosomes are needed.

  7. Visualization and quantification of archaeal and bacterial metabolically active cells in soil using fluorescence in situ hybridization method

    NASA Astrophysics Data System (ADS)

    Semenov, Mikhail; Manucharova, Natalia; Stepanov, Alexey

    2015-04-01

    The method of in situ hybridization using fluorescent labeled 16S rRNA-targeted oligonucleotide probes (FISH - fluorescence in situ hybridization) combines identification and quantification of groups of microorganisms at different phylogenetic levels, from domain to species. The FISH method enables to study the soil microbial community in situ, avoiding plating on nutrient media, and allows to identify and quantify living, metabolically active cells of Bacteria and Archaea. The full procedure consists of the following steps: desorption of the cells from the soil particles, fixation of cells, coating a fixed sample on the glass slide, hybridization with the specific probes and, finally, microscopic observation and cell counting. For the FISH analysis of Bacteria and Archaea, the paraformaldehyde-fixed samples were hybridized with Cy3-labeled Archaea-specific probe(Arc915) and 6-carboxyfluorescein (FAM)-labeled Bacteria-specific probe(EUB338). When a molecular probe is incorporated into a cell, it can hybridize solely with a complementary rRNA sequence. The hybridization can be visualized under the fluorescent microscope and counted. The application of FISH will be demonstrated by the abundance of metabolically active cells of Archaea and Bacteria depending on soil properties, depth and land use. The research was carried out at field and natural ecosystems of European part of Russia. Samples were collected within the soil profiles (3-6 horizons) of Chernozem and Kastanozem with distinct land use. Quantification of metabolically active cells in virgin and arable Chernozem revealed that the abundance of Archaea in topsoil of virgin Chernozem was doubled as compared with arable soil, but it leveled off in the deeper horizons. Plowing of Chernozem decreased an amount of archaeal and bacterial active cells simultaneously, however, Bacteria were more resistant to agrogenic impact than Archaea. In Kastanozem, a significant change in the abundance of metabolically active cells due to plowing was detected only within 40 cm soil layer, and this effect disappeared in lower horizons. The abundance of Archaea was higher in the upper horizons of arable soil as compared to virgin. Conversely, the abundance of Bacteria in the upper layers of arable Kastanozem decreased versus virgin soil. A relationship between soil organic carbon and the amount of soil metabolically active Bacteria and Archaea cells revealed that distribution of both Bacteria and Archaea throughout the soil profile was governed mostly by the organic matter content. Thus, the organic matter was a main factor of declining the Bacteria:Archaea ratio with the soil depth (from 7.1 to 4.2 for virgin soil and from 5 to 3.9 for arable soil). As a result, Archaea out-compete Bacteria under conditions of reduced energy supply. Thus, the FISH method combines classical microscopic and modern phylogenetic microbiological approaches and can be considered as an effective tool for ecological, diagnostic and environmental research in microbiology.

  8. Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: Clinical experience with 4,500 specimens

    SciTech Connect

    Ward, B.E.; Gersen, S.L.; Carelli, M.P.; McGuire, N.M.; Dackowski, W.R.; Klinger, K.W. ); Weinstein, M. ); Sandlin, C. ); Klinger, K.W. )

    1993-05-01

    Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). The authors herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies. 40 refs., 1 fig., 5 tabs.,

  9. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1, 1992--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  10. Directly incorporating fluorochromes into DNA probes by PCR increases the efficience of fluorescence in situ hybridization

    SciTech Connect

    Dittmer, Joy

    1996-05-01

    The object of this study was to produce a directly labeled whole chromosome probe in a Degenerative Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) that will identify chromosome breaks, deletions, inversions and translocations caused by radiation damage. In this study we amplified flow sorted chromosome 19 using DOP-PCR. The product was then subjected to a secondary DOP PCR amplification, After the secondary amplification the DOP-PCR product was directly labeled in a tertiary PCR reaction with rhodamine conjugated with dUTP (FluoroRed) to produce a DNA fluorescent probe. The probe was then hybridized to human metaphase lymphocytes on slides, washed and counterstained with 4{prime},6-diamino-2-phenylindole (DAPI). The signal of the FluoroRed probe was then compared to a signal of a probe labeled with biotin and stained with avidin fluorescein isothio cynate (FITC) and anti-avidin FITC. The results show that the probe labeled with FluoroRed gave signals as bright as the probe with biotin labeling. The FluoroRed probe had less noise than the biotin labeled probe. Therefore, a directly labeled probe has been successfully produced in a DOP-PCR reaction. In future a probe labeled with FluoroRed will be produced instead of a probe labeled with biotin to increase efficiency.

  11. Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

    PubMed Central

    Wang, Xiaozhu; Takebayashi, Shin-ichiro; Bernardin, Evans; Gilbert, David M.; Chella, Ravindran

    2012-01-01

    We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells. PMID:22231286

  12. Cytogenetic follow-up by karyotyping and fluorescence in situ hybridization: implications for monitoring patients with myelodysplastic syndrome and deletion 5q treated with lenalidomide

    PubMed Central

    Göhring, Gudrun; Giagounidis, Aristoteles; Büsche, Guntram; Hofmann, Winfried; Kreipe, Hans Heinrich; Fenaux, Pierre; Hellström-Lindberg, Eva; Schlegelberger, Brigitte

    2011-01-01

    In patients with low and intermediate risk myelodysplastic syndrome and deletion 5q (del(5q)) treated with lenalidomide, monitoring of cytogenetic response is mandatory, since patients without cytogenetic response have a significantly increased risk of progression. Therefore, we have reviewed cytogenetic data of 302 patients. Patients were analyzed by karyotyping and fluorescence in situ hybridization. In 85 patients, del(5q) was only detected by karyotyping. In 8 patients undergoing karyotypic evolution, the del(5q) and additional chromosomal aberrations were only detected by karyotyping. In 3 patients, del(5q) was only detected by fluorescence in situ hybridization, but not by karyotyping due to a low number of metaphases. Karyotyping was significantly more sensitive than fluorescence in situ hybridization in detecting the del(5q) clone. In conclusion, to optimize therapy control of myelodysplastic syndrome patients with del(5q) treated with lenalidomide and to identify cytogenetic non-response or progression as early as possible, fluorescence in situ hybridization alone is inadequate for evaluation. Karyotyping must be performed to optimally evaluate response. (clinicaltrials.gov identifier: NCT01099267 and NCT00179621) PMID:21109690

  13. A prospective comparative study on fluorescence in situ hybridization (FISH) of uncultured amniocytes and standard karyotype analysis.

    PubMed

    Eiben, B; Trawicki, W; Hammans, W; Goebel, R; Epplen, J T

    1998-09-01

    Fluorescence in situ hybridization (FISH) on uncultured amniocytes and standard cytogenetic analysis after amniocentesis have been performed for 904 samples. The experience with the FISH method and its clinical relevance is described in a large clinical pilot study. Commercially available chromosome-specific DNA probes for chromosomes 13, 18, 21, X and Y were used. FISH assays were performed from 12 weeks of gestation to the third trimester. In 96 per cent of the cases, hybridization was performed successfully. At least 50 nuclei for all probes could be counted in 88 per cent of the cases and in 8 per cent between 10 and 49 nuclei were scored. All trisomies 13, 18 and 21 and all cases with gonosomal aberrations were detected by FISH analysis with the exception of one case of trisomy 21 in which hybridization failed due to technical problems. Neither false-positive nor false-negative results were obtained with the DNA probes, in complete agreement with standard cytogenetics. In our experience, FISH is a valuable and reliable method for rapid diagnosis. Consequences of FISH diagnosis are discussed. PMID:9793971

  14. Assignment of the gastric inhibitory polypeptide receptor gene (GIPR) to chromosome bands 19q13.2-q13.3 by fluorescence in situ hybridization

    SciTech Connect

    Stoffel, M.; Fernald, A.A.; Bell, G.I.; Le Beau, M.M.

    1995-08-10

    The gastric inhibitory polypeptide receptor gene (GIPR) was localized, using fluorescence in situ hybridization (FISH), to human chromosome bands 19q13.2-q13.3. Gastric inhibitory polypeptide (GIP) is a potent stimulator of insulin secretion and mutations in the GIPR gene may be related to non-insulin-dependent diabetes mellitus (NIDDM). 13 refs., 1 fig.

  15. Stellaris(®) RNA Fluorescence In Situ Hybridization for the Simultaneous Detection of Immature and Mature Long Noncoding RNAs in Adherent Cells.

    PubMed

    Orjalo, Arturo V; Johansson, Hans E

    2016-01-01

    RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the detection and localization of RNA, is becoming an increasingly important complement to other gene expression analysis methods. Especially important for long noncoding RNAs (lncRNAs), RNA FISH adds the ability to distinguish between primary and mature lncRNA transcripts and thus to segregate the site of synthesis from the site of action.We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple primary and mature mRNA and lncRNA gene products and RNA variants in fixed mammalian cells. The technique makes use of fluorescently pre-labeled, short DNA oligonucleotides (circa 20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in fluorescent signals that reveal clusters of RNAs or single RNA molecules as punctate spots without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the counting of single transcripts down to one copy per cell. Additionally, by using probe sets with spectrally distinct fluorophores, multiplex analysis of gene-specific RNAs, or RNA variants, can be achieved. The presented examples illustrate how this method can add temporospatial information between the transcription event and both the location and the endurance of the mature lncRNA. We also briefly discuss post-processing of images and spot counting to demonstrate the capabilities of this method for the statistical analysis of RNA molecules per cell. This information can be utilized to determine both overall gene expression levels and cell-to-cell gene expression variation. PMID:26721487

  16. Technical Note: Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent In Situ Hybridization (FISH)

    NASA Astrophysics Data System (ADS)

    Borrelli, C.; Sabbatini, A.; Luna, G. M.; Nardelli, M. P.; Sbaffi, T.; Morigi, C.; Danovaro, R.; Negri, A.

    2011-08-01

    Benthic foraminifera are an important component of the marine biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically, these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but does not allow discrimination between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a new and useful approach to identify living cells possessing an active metabolism. Our work is the first test of the suitability of the FISH technique, based on fluorescent probes targeting the 18S rRNA, to detect live benthic foraminifera. The protocol was applied on Ammonia group and Miliolids, as well as on agglutinated polythalamous (i.e., Leptohalysis scottii and Eggerella scabra) and soft-shelled monothalamous (i.e., Psammophaga sp. and saccamminid morphotypes) taxa. The results from FISH analyses were compared with those obtained, on the same specimens assayed with FISH, from microscopic analysis of the cytoplasm colour, presence of pigments and pseudopodial activity. Our results indicate that FISH targets only metabolically active foraminifera, and allows discerning from low to high cellular activity, validating the hypothesis that the intensity of the fluorescent signal emitted by the probe is dependent upon the physiological status of cells. These findings support the usefulness of this molecular approach as a key tool for obtaining information on the physiology of living foraminifera, both in field and experimental settings.

  17. Genotype-phenotype correlation in satellited 1p chromosome: Importance of fluorescence in situ hybridization (FISH) applications

    SciTech Connect

    Habibian, R.; Hajianpour, M.J.; Hajianpour, A.K.

    1994-09-01

    Fluorescence in situ hybridization (FISH) was used to delineate the structural rearrangement of two satellited 1p chromosomes identified in a prenatal and in a postnatal case. Prenatal case: chromosome analysis of amniotic cells on a 37-year-old G2, PO, SAb1 woman, referred for advanced maternal age, revealed a 46,XX,1ps karyotype. Parental chromosome analyses showed that the satellited chromosome was paternal in origin. The satellited 1p did not stain with NOR and DA-DAPI. FISH using a probe specific for the rDNA, 18S and 28S genes also showed no hybridization to the satellited 1p. Using two different probes specific for 1p36.3 showed hybridization to both chromosomes 1p. This indicated that the terminal band was most likely present in the fetus and chromosomally balanced like the father. The pregnancy was continued and a phenotypically normal female was born. Postnatal case: Chromosome analysis of peripheral lymphocytes was performed on a 3 1/2-year old female with multiple congenital anomalies including growth retardation, developmental delay, upslanted palpebral fissures, thick eyebrows, esotropia, seizures, and mental retardation. She was born to a 14-year old, G1, PO mother, after a full-term pregnancy, by cesarian-section due to breech presentation. Her one-year-old brother is reportedly in good health. Chromosome analysis revealed a de novo 46,XX,lps. NOR and DA-DAPI stains were positive indicating the satellites are most likely chromosome 15 in origin. FISH using the rDNA probes also showed a hybridization to the 1ps. The two 1p36.3 probes showed only one signal to the normal chromosome 1, indicating a deletion which resulted in monosomy of 1p36.3. The clinical features of the child correlates with published cases of the terminal deletions of 1p.

  18. Ordering of probes surrounding the Ewing's sarcoma breakpoint on chromosome 22 using fluorescent in situ hybridization to interphase nuclei.

    PubMed

    Shipley, J M; Jones, T A; Patel, K; Kiely, F M; De Stavola, B L; Sheer, D

    1993-01-01

    Eight probes were localized by fluorescent in situ hybridization to the region surrounding the Ewing's sarcoma breakpoint on chromosome 22. Three of these were initially ordered by pair-wise hybridization to metaphase chromosomes with differential detection of the probes. These and the remaining probes were then ordered by hybridizing two or three probes simultaneously to interphase nuclei. In the two probe experiments and some of the three probe experiments, the order was derived by comparing mean interphase distances between signals from the probes. In the three probe experiments, either two probes were detected with one fluorochrome and the third with another or all three probes were individually distinguished by detecting one probe with fluorescein isothiocyanate (FITC), one with Texas Red, and one with both fluorochromes to give a mixed color. The order of the signals was then noted. Greater than 60 percent of configurations with a discrete order were shown or deduced to be correct. These approaches are assessed and we demonstrate a more obvious predominating order when all three probes are differentially detected. The order of the probes was deduced to be centromere: D22S271: D22S260: lambda S1: D22S262: cosK1831: Ewing's sarcoma breakpoint: cosLIF: D22S261: lambda S15: telomere. PMID:8404047

  19. Chromosome analysis of nuclear power plant workers using fluorescence in situ hybridization and Giemsa assay

    PubMed Central

    Hristova, Rositsa; Hadjidekova, Valeria; Grigorova, Mira; Nikolova, Teodora; Bulanova, Minka; Popova, Ljubomira; Staynova, Albena; Benova, Donka

    2013-01-01

    The aim of this study was to evaluate the genotoxic effects of ionizing radiation in vivo in exposed Bulgarian nuclear power plant workers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes. Chromosome analysis using fluorescence in situ hybrydization (FISH) and Giemsa techniques was undertaken on 63 workers and 45 administrative staff controls from the Bulgarian Nuclear Power Plant. Using the Giemsa method, the frequencies of cells studied with chromosome aberrations, dicentrics plus rings and chromosome fragments in the radiation workers were significantly higher compared with the control group (P = 0.044, P = 0.014, and P = 0.033, respectively). A significant association between frequencies of dicentrics plus rings and accumulated doses was registered (P < 0.01). In the present study, a FISH cocktail of whole chromosome paints for chromosomes 1, 4 and 11 was used. A significant association between frequency of translocations and accumulated doses was also observed (P < 0.001). Within the control group, a correlation was found between age and the spontaneous frequency of translocations. No correlation was found between smoking status and frequency of translocations. When compared with the control group, workers with accumulated doses up to 100 mSv showed no increase in genome translocation frequency, whereas workers with accumulated doses from 101 to 200 mSv showed a statistically significant doubling of genome translocation frequency (P = 0.009). Thus, in cases of chronic exposure and for purposes of retrospective dosimetry, the genome frequency of translocations is a more useful marker for evaluation of genotoxic effects than dicentric frequency. PMID:23536543

  20. Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria

    SciTech Connect

    Martin, R.H. |; Rademaker, A.

    1994-09-01

    Aneuploidy estimates for individual chromosomes in human sperm have varied more than 10-fold in different laboratories using fluorescence in situ hybridization (FISH). These laboratories use different scoring criteria in the assessment of a disomic sperm. In order to determine reliable estimates of aneuploidy, we have investigated whether scoring criteria affect the aneuploidy frequency in human sperm. Aneuploidy estimates for chromosomes 1(pUC1.77), 12(pBR12), X(XC) and Y(DYZ3Z) were obtained in human sperm from five donors using multicolor FISH analysis to provide an internal control to differentiate between nullisomy and lack of hybridization and between disomy and diploidy. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one scoring criterion used one-half a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other scoring criterion set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half domain as the scoring criterion and 113,478 were scored using one domain as the criterion. The mean percent disomy for chromosomes 1, 12, X, Y and XY was .18, .16, .15, .19, .25 respectively using the one-half domain criterion and .08, .17, .07, .12, .16 respectively using the one domain criterion. The percent disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X and Y split into more than one domain in decondensed interphase sperm and use of the one-half domain criterion leads to an overestimate of aneuploidy frequencies.

  1. Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis.

    PubMed

    Nikolakakis, K; Lehnert, E; McFall-Ngai, M J; Ruby, E G

    2015-07-01

    The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization. PMID:25956763

  2. Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization

    SciTech Connect

    Wyrobek, A.J.; Robbins, W.A. |; Pinkel, D.; Weier, H.U.; Mehraein, Y. |

    1994-10-15

    Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of {approximately} 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of {approximately} 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring. 44 refs., 1 fig., 3 tabs.

  3. Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent in situ Hybridization (FISH)

    NASA Astrophysics Data System (ADS)

    Borrelli, C.; Sabbatini, A.; Luna, G. M.; Morigi, C.; Danovaro, R.; Negri, A.

    2010-10-01

    Benthic foraminifera are an important component of the marine living biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but this approach does not allow discriminating between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a potentially useful approach identifying living cells with active metabolism cells. In this work, we tested for the first time the suitability of the FISH technique based on fluorescent probes targeting the 18S rRNA, to detect these live benthic protists. The protocol was applied on the genus Ammonia, on the Miliolidae group and an attempt was made also with agglutinated species (i.e., Leptohalysis scottii and Eggerella scabra). In addition microscopic analysis of the cytoplasm colour, presence of pigments and, sometimes, those of pseudopodial activity where conducted. The results of the present study indicate that FISH targeted only live and metabolically active foraminifera. These results allowed to identify as "live", cells improperly classified as "dead" by means of the classical technique (Type I error) and vice versa to identify as dead the foraminifera without rRNA, but stained using Rose Bengal (Type II error). In addition, the comparative FISH analysis of starved and actively growing cells demonstrated that individuals with active metabolism were stained more intensively than starved cells. This finding supports the hypothesis that the physiological status of cells can be directly related with the intensity of the fluorescent signal emitted by the fluorescent probe. We conclude that the use of molecular approaches could represent a key tool for acquiring crucial information on living foraminifera specimens and for investigating their ecological role in marine sediments.

  4. Fluorescent in situ hybridization of pre-incubated blood culture material for the rapid diagnosis of histoplasmosis.

    PubMed

    da Silva, Roberto Moreira; da Silva Neto, João Ricardo; Santos, Carla Silvana; Cruz, Kátia Santana; Frickmann, Hagen; Poppert, Sven; Koshikene, Daniela; de Souza, João Vicente Braga

    2015-02-01

    Fluorescence in situ hybridization (FISH) has been shown to be useful for the detection of Candida and Cryptococcus species in blood culture materials. FISH procedures for the detection of Histoplasma capsulatum var. capsulatum have not been reported so far. This study describes the development and evaluation of fluorescently labeled rRNA-targeting FISH probes to detect and identify H. capsulatum in blood cultures. All three analyzed H. capsulatum reference strains and clinical isolates showed positive signals with the newly designed specific oligonucleotide probes for H. capsulatum, whereas negative reactions were observed for all three nontarget yeast species and the two nontarget bacteria. The assay was also successfully applied for detections of H. capsulatum cells in pre-incubated blood culture samples of patients with clinical suspicion of histoplasmosis (n = 33). The described FISH-based assay was shown to be easy to apply, sensitive, and specific (compared to polymerase chain reaction) for the detection and identification of H. capsulatum in this proof-of-principle analysis. Larger multicentric assessments are recommended for a thorough diagnostic evaluation of the procedure. PMID:25537280

  5. Combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of Salmonella on fresh produce.

    PubMed

    Bisha, Bledar; Brehm-Stecher, Byron F

    2010-01-01

    This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55°C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (? 500 ?L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37°C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalapeño peppers. PMID:21048665

  6. The human sorbitol dehydrogenase gene: cDNA cloning, sequence determination, and mapping by fluorescence in situ hybridization

    SciTech Connect

    Lee, F.K.; Chung, S. ); Cheung, M.C. )

    1994-05-15

    The cDNA for human sorbitol dehydrogenase (SORD) has been cloned and sequenced. It translates into a peptide of 356 amino acid residues, one more than the sequence previously reported from peptide analysis. An extra alanine was found at the acetyl-blocked N-terminal, between positions 1 and 4. This matches the rat cDNA, which also has 356 amino acids, with an extra proline at position 3. Four other mismatches were also observed, but these are all amino acid substitutions that occur outside proposed functionally important regions. Further work must be performed to determine whether these discrepancies represent polymorphic forms of the enzyme. The SORD gene was mapped by fluorescence in situ hybridization and found to occupy a single site on chromosome 15q15, indicating that it is a single-copy gene. This was confirmed by Southern blot hybridization. SORD is thought to be involved in the etiology of diabetic complications, and its deficiency has been linked to congenital cataracts. The cloned gene could be used as a probe to study the role of this enzyme in the pathogenesis of these diseases. 24 refs., 4 figs.

  7. Assignment of the human aggrecan gene (AGC1) to 15q26 using fluorescence in situ hybridization analysis

    SciTech Connect

    Korenberg, J.R.; Chen, X.N.; Doege, K.; Grover, J.; Roughley, P.J.

    1993-05-01

    The large aggregating proteoglycan aggrecan is a major structural component of the extracellular matrix of articular cartilage. Recent cDNA cloning of the human aggrecan gene (AGC1) reveals a core protein of at least 2316 amino acids characterized by several distinct structural domains. Two globular domains, termed G1 and G2, are present at the amino terminus of the molecule and a third, termed G3, is present at the carboxy terminus. The G1 domain is homologous in structure to the cartilage link protein and accounts for the aggregating potential of aggrecan through its ability to interact with hyaluronic acid. The aggrecan gene is known to consist of 15 exons, with each exon encoding a distinct functional region of the mature protein. However, while the link protein gene is known to reside on chromosome 5 in the human, the location of the aggrecan gene is currently undetermined in any species. The probe (pAGG2) for the aggrecan gene was mapped on chromosome band 15q26, most likely in the subregion of 15q26.1, using fluorescence in situ hybridization. Clear signals were noted on both chromatids of chromosome band 15q26 in over 80% of the 300 metaphase cells examined in three independent experiments using pAGG2. No other sites of hybridization were noted on both chromatids of any other chromosome band. The precise band location was identified by using chromsomes of about 650 bands and employing fluorescence reverse banding with chromomycin A3 and distamycin. 14 refs., 1 fig.

  8. Detection of aneuploid human sperm by fluorescence in situ hybridization: Evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y

    SciTech Connect

    Robbins, W.A. Lawrence Livermore National Lab., CA ); Segraves, R.; Pinkel, D. ); Wyrobek, A.J. )

    1993-04-01

    Fluorescence in situ hybridization with repetitive-sequence DNA probes was used to detect human sperm disomic for chromosomes 1 and Y in three healthy men. Data on these same men had been obtained previously, using the human-sperm/hamster-egg cytogenetic technique, providing a cytogenetic reference for validating sperm hybridization measurements. Air-dried smears were prepared from semen samples and treated with DTT and lithium diiodosalicylate to expand sperm chromatin. Hybridization with fluorescently tagged DNA probes for chromosomes 1 (pUC177) or Y (pY3.4) yielded average frequencies of sperm with two fluorescent domains of 14.2[+-]2.4/10,000 and 5.6[+-]1.6/10,000 sperm, respectively. These frequencies did not differ statistically from frequencies of hyperploidy observed for these chromosomes with the hamster technique. In addition, frequencies of disomic sperm from one donor were elevated [approximately]2.5-fold above those of other donors, for both chromosomes 1 (P = .045) and Y (P = .01), consistent with a trend found with the hamster technique. The authors conclude that fluorescence in situ hybridization to sperm chromosomes provides a valid and promising measure of the frequency of disomic human sperm. 43 refs., 1 fig., 4 tabs.

  9. Aneuploidy detection for chromosomes 1, X and Y by fluorescence in situ hybridization in human sperm from oligoasthenoteratozoospermic patients

    SciTech Connect

    Pang, M.G.; Zackowski, J.L.; Acosta, A.A.

    1994-09-01

    Oligoasthenoteratozoospermic males (n=15) were investigated for infertility as compared with proven fertile donors. The oligoasthenoteratozoospermic population showed a mean sperm concentration of 9.7 x 10{sup 6}/ml (Range 4.2-19.7), mean motility of 38.5% (Range 10.6-76.8) and morphology (measured by the percentage of normal forms evaluated by strict criteria) with a mean of 3.49% (Range 1.5-5.0). Fluorescence in situ hybridization (FISH) using satellite DNA probes specific for chromosomes 1 (puc 1.77), X (alpha satellite), and Y (satellite-III at Yqh) was performed on human interphase sperm nuclei. DNA probes were either directly labelled with rhodamine-dUTP, FITC-dUTP, or biotinylated by nick translation. Hybridization and signal detection were done by routine laboratory protocols. Microscopic analysis was performed using a cooled CCD camera attached to an epi-fluorescent microscope. After hybridization, fertile donors yielded a frequency of 0.96% (n=12) nullisomic, 98.5% (n=1231) monosomic and 0.96% (n=12) disomic for chromosome 1, whereas oligoasthenoteratozoospermic males yielded a frequency of 16% (n=600) nullisomic, 74.5% (n=2792) monosomic and 9.9% (n=370) disomic. In addition, fertile donors yielded a frequency of 45.7% (n=633) monosomic and 0.7% (n=11) disomic for chromosome X, whereas oligoasthenoteratozoospermic males yielded a frequency of 38.7% (n=760) monosomic and 0.8% (n=13) disomic. Chromosome Y frequencies for fertile donors showed 44.6% (n=614) monosomic and 0.6% (n=2) disomic, whereas oligoasthenoteratozoospermic males yielded a frequency of 33.2% (n=701) monosomic and 0.8% (n=15) disomic. This suggests that the frequency of nullisomy for chromosome 1 is significantly higher (p<0.001) in sperm from oligoasthenoteratozoospermic makes versus sperm from our fertile donors. We conclude that FISH is a powerful tool to determine the frequency of aneuploidy in sperm from oligoasthenoteratozoospermic patients.

  10. Paternal-age effects on sperm aneuploidy investigated in mice and humans by three-chromosome fluorescence in situ hybridization

    SciTech Connect

    Wyrobek, A.J.; Lowe, X.; Holland, N.T.

    1994-09-01

    We conducted a cross-species comparison of the effects of paternal age on sperm aneuploidy in mice and humans. A new murine assay was developed to detect sperm hyperhaploidy and polyploidy for chromosomes X, Y, and 8 using fluorescence in situ hybridization with chromosome-specific DNA probes, to serve as a direct corollate to the three-chromosome method developed early for human sperm. Sperm aneuploidy was evaluated in eight male B6C3F1 male mice (aged 22.5-30.5 mo) and compared to young controls (2.4 mo). The aged group showed significant ({approximately}2.0-fold) increases in hyperhaploidies involving chromosomes X, Y and 8, with the greatest effects seen in the oldest animals. Sperm aneuploidy was also evaluated in two groups of healthy men who differed in mean age [46.8{plus_minus}3.1 (n=4) vs. 28.5{plus_minus}5.0 (n=10) yrs], using the three-chromosome method. The older group showed a statistically significant increase in hyperhaploid sperm for both sex chromosomes. Additional controlled human studies are planned. Taken together, the murine and human data are consistent with a positive effect of paternal age on sperm aneuploidy. In both species, the strongest age effect was observed for hyperhaploidies of chromosome Y. Future studies are needed to investigate the shape of the age-effect curve and to evaluate chromosomal differences, especially for humans in their late reproductive years.

  11. Fluorescence in situ hybridization of TP53 for the detection of chromosome 17 abnormalities in myelodysplastic syndromes.

    PubMed

    Sánchez-Castro, Judit; Marco-Betés, Víctor; Gómez-Arbonés, Xavier; García-Cerecedo, Tomás; López, Ricard; Talavera, Elisabeth; Fernández-Ruiz, Sara; Ademà, Vera; Marugan, Isabel; Luño, Elisa; Sanzo, Carmen; Vallespí, Teresa; Arenillas, Leonor; Marco Buades, Josefa; Batlle, Ana; Buño, Ismael; Martín Ramos, María Luisa; Blázquez Rios, Beatriz; Collado Nieto, Rosa; Vargas, Ma Teresa; González Martínez, Teresa; Sanz, Guillermo; Solé, Francesc

    2015-11-01

    Conventional G-banding cytogenetics (CC) detects chromosome 17 (chr17) abnormalities in 2% of patients with de novo myelodysplastic syndromes (MDS). We used CC and fluorescence in situ hybridization (FISH) (LSI p53/17p13.1) to assess deletion of 17p in 531 patients with de novo MDS from the Spanish Group of Hematological Cytogenetics. FISH detected - 17 or 17p abnormalities in 13 cases (2.6%) in whom no 17p abnormalities were revealed by CC: 0.9% of patients with a normal karyotype, 0% in non-informative cytogenetics, 50% of patients with a chr17 abnormality without loss of 17p and 4.7% of cases with an abnormal karyotype not involving chr17. Our results suggest that applying FISH of 17p13 to identify the number of copies of the TP53 gene could be beneficial in patients with a complex karyotype. We recommend using FISH of 17p13 in young patients with a normal karyotype or non-informative cytogenetics, and always in isolated del(17p). PMID:25754580

  12. Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms.

    PubMed Central

    Poulsen, L K; Ballard, G; Stahl, D A

    1993-01-01

    We describe the in situ use of rRNA-targeted fluorescent hybridization probes in combination with digital microscopy to quantify the cellular content of ribosomes in relationship to the growth rate of single cells of a specific population of sulfate-reducing bacteria in multispecies anaerobic biofilms. Using this technique, we inferred that this population was growing with an average generation time of 35 h in a young biofilm, whereas the doubling time in an established biofilm was significantly longer. Conventional chemical determinations of the RNA, DNA, and protein contents of this culture at different growth rates were also carried out, and the resulting data were compared with the rRNA fluorescence in situ hybridization data. Images PMID:7685999

  13. Microbial populations identified by fluorescence in situ hybridization in a constructed wetland treating acid coal mine drainage

    SciTech Connect

    Nicomrat, D.; Dick, W.A.; Tuovinen, O.H.

    2006-07-15

    Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised of Fe(III) precipitates, were collected from two treatment cells of the constructed wetland system. The pH of the bulk samples ranged between pH 2.1 and 3.9. Viable counts of acidophilic Fe and S oxidizers and heterotrophs were determined with a most probable number (MPN) method. The MPN counts were only a fraction of the corresponding FISH counts. The sediment samples contained microorganisms in the Bacteria (including the subgroups of acidophilic Fe- and S-oxidizing bacteria and Acidiphilium spp.) and Eukarya domains. Archaea were present in the sediment surface samples at < 0.01% of the total microbial community. The most numerous bacterial species in this wetland system was Acidithiobacillus ferrooxidans, comprising up to 37% of the bacterial population. Acidithiobacillus thiooxidans was also abundant.

  14. Triplex in-situ hybridization

    SciTech Connect

    Fresco, Jacques R.; Johnson, Marion D.

    2002-01-01

    Disclosed are methods for detecting in situ the presence of a target sequence in a substantially double-stranded nucleic acid segment, which comprises: a) contacting in situ under conditions suitable for hybridization a substantially double-stranded nucleic acid segment with a detectable third strand, said third strand being capable of hybridizing to at least a portion of the target sequence to form a triple-stranded structure, if said target sequence is present; and b) detecting whether hybridization between the third strand and the target sequence has occured.

  15. Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization.

    PubMed

    Selinger, Christina I; Rogers, Toni-Maree; Russell, Prudence A; O'Toole, Sandra; Yip, Poyee; Wright, Gavin M; Wainer, Zoe; Horvath, Lisa G; Boyer, Michael; McCaughan, Brian; Kohonen-Corish, Maija Rj; Fox, Stephen; Cooper, Wendy A; Solomon, Benjamin

    2013-12-01

    Rearrangements of anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) define a molecular subgroup of tumors characterized clinically by sensitivity to ALK tyrosine kinase inhibitors such as crizotinib. Although ALK rearrangements may be detected by reverse transcriptase-PCR, immunohistochemistry or fluorescence in situ hybridization (FISH), the optimal clinical strategy for identifying ALK rearrangements in clinical samples remains to be determined. We evaluated immunohistochemistry using three different antibodies (ALK1, 5A4 and D5F3 clones) to detect ALK rearrangements and compared those with FISH. We report the frequency and clinicopathologic features of lung cancers harboring ALK translocations in 594 resected NSCLCs (470 adenocarcinomas; 83 squamous carcinomas, 26 large cell carcinomas and 15 other histological subtypes) using a tissue microarray approach. We identified an ALK gene rearrangement in 7/594 cases (1%) by FISH and all anti-ALK antibodies correctly identified the seven ALK-positive cases (100% sensitivity), although the intensity of staining was weak in some cases. These data indicate that the use of antibodies with high sensitivity and avidity to ALK may provide an effective pre-screening technique to complement the more expensive and labor-intensive approach of ALK FISH testing. PMID:23743928

  16. Monitoring of chimerism using fluorescence in situ hybridization in a child with severe combined immune deficiency following bone marrow transplant

    SciTech Connect

    Wenger, S.L.; Chen, X.O.; Katz, A.J. |

    1994-09-01

    A boy with severe combined immunodeficiency received a bone marrow transplant from his sister when he was approximately 3 years of age. His peripheral blood karyotype at age 3 and 4 years was 46,XX (20 cells analyzed). Because of a decline in antibody production at 19 years of age, the patient`s peripheral blood was analyzed again for suspected chimerism. His karyotype in phytohemagglutinin (PHA)-stimulated culture was 46,XX in 49 cells and 46,XY in one cell. Both metaphase and interphase cells were examined for sex chromosome constitution using X and Y dual-color alpha-satellite probes for fluorescence in situ hybridization (FISH). FISH results for metaphase cells showed 1/50 XY cells, but 38% of interphase cells showed the presence of both X and Y centromere. Pokeweed mitogen (PWM)-stimulated cultures grew poorly and were therefore analyzed using FISH only: 81% of interphase cells were 46,XX. The discrepancy between metaphase and interphase in the PHA-stimulated cultures most likely represents a failure of this boy`s own XY T-cells to be stimulated.

  17. A Novel 4-color Fluorescence in Situ Hybridization Assay for Detection of TMPRSS2 and ERG Rearrangements in Prostate Cancer

    PubMed Central

    Qu, Xiaoyu; Randhawa, Grace; Friedman, Cynthia; O'Hara-Larrivee, Siobhan; Kroeger, Kathleen; Dumpit, Ruth; True, Larry; Vakar-Lopez, Funda; Porter, Christopher; Vessella, Robert; Nelson, Peter; Fang, Min

    2013-01-01

    Purpose Since the identification of TMPRSS2/ERG rearrangement as the most common fusion event in prostate cancer, various methods have been developed to detect this rearrangement and to study its prognostic significance. We hereby report a novel 4-color fluorescence in situ hybridization (FISH) assay that not only detects the typical TMPRSS2:ERG fusion but also alternative rearrangements of either the TMPRSS2 or ERG gene. Experimental design We validated this assay on fresh, frozen, or formalin-fixed paraffin-embedded prostate cancer specimens including cell lines, primary prostate cancer, xenograft tissues derived from metastatic prostate cancer, and metastatic tissues from castration-resistant prostate cancer (CRPC) patients. Results When compared with RT-PCR or Gen-Probe method as the technical reference, the 4-color FISH assay demonstrated an analytical sensitivity of 94.5% (95% Confidence Interval [CI] 0.80-0.99) and specificity of 100% (95% CI 0.89-1.00) for detecting TMPRSS2:ERG fusion. TMPRSS2:ERG fusion was detected at 41% and 43% in primary prostate cancer (n = 59) and CRPC tumors (n = 82), respectively. Alternative rearrangements other than the typical TMPRSS2:ERG fusion were confirmed by karyotype analysis and shown present in 7% primary cancer and 13% CRPC tumors. Successful karyotype analysis is reported for the first time on four of the xenograft samples, complementing the FISH results. Conclusions This 4-color FISH assay provides sensitive detection of TMPRSS2 and ERG gene rearrangements in prostate cancer. PMID:23352841

  18. Quantitative Fluorescence In Situ Hybridization Analysis of Microbial Consortia from a Biogenic Gas Field in Alaska's Cook Inlet Basin

    PubMed Central

    Str?po?, Dariusz; Huizinga, Brad; Lidstrom, Ulrika; Ashby, Matt; Macalady, Jennifer L.

    2012-01-01

    Filter-collected production water samples from a methane-rich gas field in the Cook Inlet basin of Alaska were investigated using whole-cell rRNA-targeted fluorescence in situ hybridization (FISH) and 16S rRNA tag pyrosequencing. Both techniques were consistent in determining the microbial community composition, including the archaeal or bacterial dominance of samples. The archaeal community is dominated by the obligate methylotrophic methanogen genus Methanolobus as well as the nutritional generalist methanogen genus Methanosarcina, which is capable of utilizing acetate, CO2, and methyl-bearing compounds. The most-abundant bacterial groups are Firmicutes, notably of the Acetobacterium genus, and Cytophaga-Flexibacter-Bacteroides species (CFBs) affiliated with the order Bacteroidales. We observed spatial variation among samples in both the percentage of members of Archaea compared to that of members of Bacteria and the dominant members of the bacterial community, differences which could not be explained with the available geochemical data. Based upon the microbial community composition and the isotopic signature of methane associated with the Cook Inlet basin site, we propose a simplified reaction network beginning with the breakdown of coal macromolecules, followed by fermentation and methylotrophic and acetoclastic methane production. PMID:22427501

  19. A fluorescence in situ hybridization (FISH) microfluidic platform for detection of HER2 amplification in cancer cells.

    PubMed

    Kao, Kai-Jie; Tai, Chien-Hsuan; Chang, Wen-Hsin; Yeh, Ta-Sen; Chen, Tse-Ching; Lee, Gwo-Bin

    2015-07-15

    Over-expression/amplification of human epidermal growth factor receptors 2 (HER2) is a verified therapeutic biomarker for breast and gastric cancers. HER2 is also served as prognostic biomarker for gastric cancer because HER2 over-expression is associated with a 5-10% increase in cancer related death of gastric cancer. Cancer patients exhibiting HER2 over-expression can significantly improve their overall survival rates by taking the targeting drug Herceptin, which directly targets HER2. However, Herceptin has limited functions toward patients without HER2 over-expression and therefore it needs a highly specific and accurate detection method for diagnosis of HER2 over-expression. Currently, fluorescence in situ hybridization (FISH) technique is routinely employed to detect HER2 amplification. However, it is a labor-intensive, time-consuming hybridization process and is relatively costly. Furthermore, well-trained personnel are required to operate the delicate and complicate process. More importantly, it may take 1-2 days for well-trained personnel to perform a whole FISH assay. Given these limitations, we developed a new, integrated microfluidic FISH system capable of automating the entire FISH protocol which could be performed within a shorter period of time when compared to traditional methods. The microfluidic FISH chip consisted of a microfluidic control module for transportation of small amounts of fluids and a hybridization module to perform the hybridization of DNA probes and cells/tissue samples. With this approach, the new microfluidic chip was capable of performing the whole FISH assay within 20h. Four cell lines, two for non-HER2 amplification and two for HER2 amplification, and two clinical tissue samples, one for non-HER2 amplification and another for HER2 amplification, were used for verifications of the developed chip. Experimental data showed that there was no significant difference between the benchtop protocol and the chip-based protocol. Furthermore, the reagent consumption was greatly reduced (?70% reduction). Especially, only 2-?l usage for FISH deoxyribonucleic acid (DNA) probe was used, which is five-fold reduction when compared with the traditional method. It is the first time that the entire FISH assay could be automated on a single chip by using tissue samples. The microfluidic system developed herein is therefore promising for rapid, automatic diagnosis of HER2-related diseases by detecting the HER2 gene with minimal consumption of samples and reagents and has a great potential for future pharmacogenetic diagnostics and therapy. PMID:25770459

  20. Application of fluorescence in situ hybridization for the study and characterization of nitrifying bacteria in nitrifying/denitrifying wastewater treatment plants.

    PubMed

    Benakova, A; Wanner, J

    2013-01-01

    The aim of this study was to verify fluorescence in situ hybridization for the detection of nitrifying bacteria in activated sludge and biofilms, and to determine the distribution of nitrifiers in selected wastewater treatment plants (WWTPs). Both Czech and foreign WWTPs with intensification of nitrification (for example, in situ bioaugmentation of nitrification or biofilms) and without intensification were studied. The two-dimensional and three-dimensional analyses of microscopic images were focused on quantifying the parameters and their differences with regard to the arrangement, capacity and sludge age of the WWTPs. This is the first time such a study has been performed in the Czech Republic. PMID:24350498

  1. Comparative cytogenetics of six Indo-Pacific moray eels (Anguilliformes: Muraenidae) by chromosomal banding and fluorescence in situ hybridization.

    PubMed

    Coluccia, E; Deidda, F; Cannas, R; Lobina, C; Cuccu, D; Deiana, A M; Salvadori, S

    2015-09-01

    A comparative cytogenetic analysis, using both conventional staining techniques and fluorescence in situ hybridization, of six Indo-Pacific moray eels from three different genera (Gymnothorax fimbriatus, Gymnothorax flavimarginatus, Gymnothorax javanicus, Gymnothorax undulatus, Echidna nebulosa and Gymnomuraena zebra), was carried out to investigate the chromosomal differentiation in the family Muraenidae. Four species displayed a diploid chromosome number 2n = 42, which is common among the Muraenidae. Two other species, G. javanicus and G. flavimarginatus, were characterized by different chromosome numbers (2n = 40 and 2n = 36). For most species, a large amount of constitutive heterochromatin was detected in the chromosomes, with species-specific C-banding patterns that enabled pairing of the homologous chromosomes. In all species, the major ribosomal genes were localized in the guanine-cytosine-rich region of one chromosome pair, but in different chromosomal locations. The (TTAGGG)n telomeric sequences were mapped onto chromosomal ends in all muraenid species studied. The comparison of the results derived from this study with those available in the literature confirms a substantial conservation of the diploid chromosome number in the Muraenidae and supports the hypothesis that rearrangements have occurred that have diversified their karyotypes. Furthermore, the finding of two species with different diploid chromosome numbers suggests that additional chromosomal rearrangements, such as Robertsonian fusions, have occurred in the karyotype evolution of the Muraenidae. PMID:26242690

  2. Single-Gene Detection and Karyotyping Using Small-Target Fluorescence in Situ Hybridization on Maize Somatic Chromosomes

    PubMed Central

    Lamb, Jonathan C.; Danilova, Tatiana; Bauer, Matthew J.; Meyer, Julie M.; Holland, Jennifer J.; Jensen, Michael D.; Birchler, James A.

    2007-01-01

    Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides. PMID:17237520

  3. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-12-31

    The ultimate goal of this proposal is to create a cDNA map of the human genome. Mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach will generate 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  4. Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

    SciTech Connect

    Worley, K.C.; Lindsay, E.A.; McCabe, E.R.B.

    1995-07-17

    Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring time-consuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosome deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. 17 refs., 2 figs.

  5. Interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction as a diagnostic aid for synovial sarcoma.

    PubMed Central

    Shipley, J.; Crew, J.; Birdsall, S.; Gill, S.; Clark, J.; Fisher, C.; Kelsey, A.; Nojima, T.; Sonobe, H.; Cooper, C.; Gusterson, B.

    1996-01-01

    Identification of the t(X;18)(p11.2;q11.2) that is associated with a high proportion of synovial sarcoma can be a useful diagnostic aid. The translocation results in fusion of the SYT gene on chromosome 18 to either the SSX1 or the SSX2 gene, two homologous genes within Xp11.2. Two-color interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction were assessed as approaches to identify the rearrangement in well characterized cases. The presence of the translocation, and the specific chromosome X gene disrupted, were inferred from the configuration of signals from chromosome-specific centromere probes, paints, and markers flanking each gene in preparations of interphase nuclei. Rearrangement was found in two cell lines and eight of nine tumor samples, including analysis of five touch imprints. This was consistent with cytogenetic data in four cases and reverse transcription polymerase chain reaction analysis using primers known to amplify both SYT-SSX1 and SYT-SSX2 transcripts. The transcripts were distinguished by restriction with LspI and SmaI. Contrary to previous suggestions, there was no obvious correlation between histological subtype and involvement of the SSX1 or SSX2 gene. These approaches could also be applied to the identification of tumor-free margins and metastatic disease. Images Figure 1 Figure 3 PMID:8579118

  6. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    NASA Astrophysics Data System (ADS)

    Tang, Xianghai; Yu, Rencheng; Zhou, Mingjiang; Yu, Zhigang

    2012-03-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

  7. Fluorescent in situ hybridization (FISH) and high resolution karyotype analysis reveal a novel inversion duplication of 10q

    SciTech Connect

    Czarnecki, P.; Dyke, D.L. Van; Dowling, P.K.

    1994-09-01

    A white male born with dysmorphic features, including upslanting palpebral fissures, bilateral simian creases, posteriorly rotated ears, bitemporal narrowing, frontal bossing, camptodactyly and head circumference and weight less than the 5th percentile was found to have a de novo add(10)(q26.1). High resolution karyotype analysis revealed a novel chromosomal abnormality: 46,XY,inv dup(10)(q26.3-q25.1). Fluorescent in situ hybridization using a chromosome 10-specific painting probe (Oncor, Inc.) confirmed that the extra material was derived from chromosome 10. Duplication of 10q24 or 10q25 is associated with characteristic craniofacial malformations, minor malformations of the hands and feet, major malformations of the heart, skeleton, and kidneys and severe mental retardation. Our patient, currently 7 months old, has many of the skeletal and craniofacial manifestations of other patients, but is developmentally normal at this early age. This is the first FISH confirmation of a 10q duplication and demonstrates the utility of this technology in addition to karyotype analysis. Molecular studies to determine the parental origin and extent of the duplication are in progress, since the apparent lack of developmental delay was unexpected. Identification of the origin of duplicated material will help assist in genetic counseling by further delineating new genetic syndromes.

  8. Quantitative chromosome map of the polyploid Saccharum spontaneum by multicolor fluorescence in situ hybridization and imaging methods.

    PubMed

    Ha, S; Moore, P H; Heinz, D; Kato, S; Ohmido, N; Fukui, K

    1999-04-01

    Somatic chromosomes of a wild relative of sugarcane (Saccharum spontaneum L.) anther culture-derived clone (AP 85-361, 2n = 32) were identified and characterized by computer-aided imaging technology and molecular cytological methods. The presence of four satellite chromosomes and four nearly identical chromosome sets suggests that the clone is a tetrahaploid with the basic number x = 8. A quantitative chromosome map, or idiogram, was developed using image analysis of the condensation pattern (CP) at the prometaphase stage of somatic chromosomes. The 45S and 5S ribosomal RNA gene (rDNA) loci were simultaneously visualized by multi-color fluorescence in situ hybridization (McFISH) and precisely localized to the regions of 3p3.1 and 6q1.3 on the idiogram. The simultaneous visualization of two sets of four ribosomal RNA genes confirms tetraploidy of this clone. This conclusion is consistent with results of molecular marker mapping. The quantitative chromosome map produced will become the foundation for genome analyses based on chromosome identity and structure. Previously impossible identification of small chromosomes and untestable hypotheses about the polyploid nature of plants can now be settled with these two approaches of quantitative karyotyping and FISH. PMID:10380803

  9. MYC Analysis by Fluorescent In Situ Hybridization and Immunohistochemistry in Primary Adrenal Angiosarcoma (PAA): a Series of Four Cases.

    PubMed

    Cornejo, Kristine M; Hutchinson, Lloyd; Cyr, Maryann St; Nose, Vania; McLaughlin, Patrick J; Iafrate, A John; Sadow, Peter M

    2015-12-01

    Primary adrenal angiosarcomas (PAA) are rare with 36 cases reported in the English literature. MYC protein expression and gene amplification have been detected in secondary angiosarcoma (AS), and a subset of primary AS. The aim of this study was to report the clinicopathologic features of PAA and examine these tumors for MYC amplification and protein expression in a small series of four cases (resection, n?=?4). Three had available material for ancillary studies and were investigated for MYC gene abnormalities and protein expression using fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Tumors occurred in three females and one male with a mean age of 69 (53-75) years. The sizes ranged from 8.5 to 15 (mean 11.5) cm and were epithelioid in morphology. All tumors had prominent necrosis, and the mitotic count ranged from 4 to 41/10 high-power fields (HPFs) (mean 20/10 HPFs, ×400). Immunohistochemically, the tumor cells were positive for CD31 in 4/4 cases, CD34 in 1/4 cases, and cytokeratin in 4/4 cases. The mean follow-up period was 10.8 (3-19) months, of which three patients died of disease with distant metastases, and one patient was alive with disease. MYC nuclear staining was identified in the three cases tested. Two cases showed polysomy of chromosome 8 without MYC amplification or rearrangement. Two MYC-positive cases by IHC demonstrated copy number gain in chromosome 8, and one MYC-positive case was not associated with a chromosome 8/MYC gene abnormality. In the context of new targeted therapies, MYC positivity in PAA may be clinically valuable in treating patients with these aggressive neoplasms. PMID:26223194

  10. Quantification of epithelial cell micronuclei by fluorescence in situ hybridization (FISH) in mortuary science students exposed to formaldehyde.

    PubMed

    Titenko-Holland, N; Levine, A J; Smith, M T; Quintana, P J; Boeniger, M; Hayes, R; Suruda, A; Schulte, P

    1996-12-20

    A micronucleus assay employing fluorescence in situ hybridization (FISH) with a centromeric probe was used on specimens of exfoliated buccal and nasal cells collected from mortuary science students exposed to embalming fluid containing formaldehyde. FISH labeling allowed micronuclei (MN) containing a whole chromosome (centromere-positive, MN+) to be differentiated from those containing only chromosomal fragments (centromere-negative, MN-). Each student was sampled before and after the 90 day embalming class. We determined if an increase in MN frequency could be attributed to formaldehyde exposure and was specific to either MN+ or MN-. In buccal cells, total MN frequency was significantly increased from 0.6/1000 to 2/1000 (p = 0.007) following the course, whereas in nasal cells it was not (2 and 2.5/1000, respectively, p = 0.2). Cells with multiple MN were present only in samples taken after exposure to embalming fluid. Although the baseline frequency was higher for MN+ in both buccal (0.4/1000 for MN+ and 0.1/1000 for MN-) and nasal cells (1.2/1000 for MN+ and 0.5/1000 for MN-), the increase in MN frequency was greater for MN-, (9-fold, p = 0.005 for buccal cells; 2-fold, p = 0.03 for nasal cells) than for MN+ (> 2-fold, p = 0.08 for buccal cells; no change, p = 0.31 for nasal cells) in both tissues. Thus, the primary mechanism of micronucleus formation appeared to be chromosome breakage. This finding is consistent with known clastogenic properties of formaldehyde, the component of embalming fluid most likely responsible for micronucleus induction. PMID:9008725

  11. Assessment of chromosomal abnormalities in sperm of infertile men using sperm karyotyping and multicolour fluorescence in situ hybridization (FISH)

    SciTech Connect

    Moosani, N.; Martin, R.H.

    1994-09-01

    Individuals with male factor infertility resulting from idiopathic oligo-, astheno- or teratozoospermia are frequently offered IVF in an attempt to increase their chances of having a child. A concern remains whether these infertile males have an elevated risk of transmitting chromosomal abnormalities to their offspring. Sperm chromosomal complements from these men were assayed using the human sperm/hamster oocyte fusion system and fluorescence in situ hybridization (FISH) on sperm nuclei. For each of 5 infertile patients, 100 sperm karyotypes were analyzed and multicolour FISH analysis was performed on a minimum of 10,000 sperm nuclei for each chromosome-specific DNA probe for chromosomes 1 (pUC1.77), 12 (D12Z3), X (XC) and Y (DYZ3). As a group, the infertile patients showed increased frequencies of both numerical ({chi}{sup 2}=17.26, {proportional_to} <0.001) and total abnormalities ({chi}{sup 2}=7.78, {proportional_to} <0.01) relative to control donors when assessed by sperm karyotypes. Analysis of sperm nuclei by FISH indicated a significant increase in the frequency of disomy for chromosome 1 in three of the five patients as compared to control donors ({chi}{sup 2}>8.35, {proportional_to} <0.005). In addition, the frequency of XY disomy was significantly higher in four of the five patients studied by FISH ({chi}{sup 2}>10.58, {proportional_to}<0.005), suggesting that mis-segregation caused by the failure of the XY bivalent to pair may play a role in idiopathic male infertility.

  12. A combination of micronucleus assay and fluorescence in situ hybridization analysis to evaluate the genotoxicity of formaldehyde.

    PubMed

    Bouraoui, Sana; Mougou, Soumaya; Brahem, Aicha; Tabka, Faten; Ben Khelifa, Hela; Harrabi, Imed; Mrizek, Najib; Elghezal, Hatem; Saad, Ali

    2013-02-01

    A genotoxic effect of formaldehyde (FA), particularly micronucleus (MN) induction, has been shown in several previous studies. The aim of the present study was to assess the frequency of micronuclei and to identify the type of chromosomal damage in Tunisian staff members working in the Pathologic Anatomy Laboratory of Farhat Hached hospital (Sousse, Tunisia) who were exposed to FA. Assessment of chromosomal damage was performed in peripheral lymphocytes of 31 FA-exposed employees compared with 31 control employees working in the administrative department of the same hospital. The clastogenic/aneugenic effect of FA was evaluated using the standard MN assay in combination with fluorescence in situ hybridization (FISH) using pan-centromeric probes. The mean level of exposure to FA was 3.4 ppm. The results showed a significant increase of MN frequency in lymphocytes of exposed workers compared with the control group (25.35 ± 6.28 ‰ vs. 7.08  ± 4.62 ‰, p < 0.05). As assessed by FISH, the frequency of centromeric micronuclei (C+MN) was greater in exposed subjects than in controls (18.38 ± 5.94 ‰ vs. 5.03 ± 3.64 ‰). Among the C+MN, the frequency of MN containing one centromere (C1+MN) was significantly greater in pathologists and anatomists than in controls (15.35 ± 6.0 ‰ vs. 3.33 ± 2.74 ‰, p < 0.05). The results showed an effect of sex and time of FA exposure with significantly increased frequencies of all end points measuring aneuploidy (C+MN, C1+MN, and Cx+MN [more then one MN]). The increased frequency of C1+MN observed in the exposed group may suggest a slight aneugenic effect of FA exposure. PMID:23132144

  13. Trisomy 10p resulting from an inv dup of 10p defined by fluorescence in situ hybridization

    SciTech Connect

    Clement, S.J.; Easterling, T.R.; Leppig, K.A.

    1994-09-01

    De novo cases of trisomy for the entire short arm of chromosome 10 are infrequently reported and are most commonly the result of translocation of 10p to an acrocentric chromosome. Most reported cases of trisomy 10p are not trisomy for the complete short arm of chromosome 10, but are duplication, deficiency syndromes that result from either inheritance of an unbalanced translocation from a parent possessing a balanced reciprocal translocation, or from a recombinant chromosome derived from a parental pericentric inversion of chromosome 10. Here, we report a case of a de novo trisomy 10p that resulted from an inverted duplication of the entire short arm of chromosome 10. A 42 year old G7,P5,SAB1 woman was referred for amniocentesis because of advanced maternal age. Ultrasound examination at 17 weeks demonstrated a fetus of normal size with no apparent anatomic abnormalities. Cytogenetic evaluation demonstrated one homologue of chromosome 10 had a tandem inverted duplication of the short arm. The fetal karyotype was interpreted to be 46,XX,inv dup (10) (peter-cen::cen-p15::q11-pter). Parental karyotype are normal. Fluorescence in situ hybridization (FISH) using a chromosome 10 paint, chromosome 10 centromere, and all telomere probe, confirmed the inverted duplication involved the entire short arm of chromosome 10. Termination of pregnancy was performed at 20 weeks gestation. Autopsy revealed multiple anomalies including low-set posteriorly rotated ears, cleft of the soft palate, ocular hypertelorism, small upturned nose, agenesis of the gallbladder, sacral hemivertebrae, and abnormal flexion of the thumbs. The fetal karyotype was confirmed by cytogenetic analysis in lung and kidney. This is the second reported case of a de novo tandem duplication of 10p of which we are aware, and the first using FISH technology to characterize the abnormality.

  14. IDENTIFICATION OF SEX CHROMOSOME MOLECULAR MARKERS USING RAPDS AND FLUORESCENT IN SITU HYBRIDIZATION IN RAINBOW TROUT

    EPA Science Inventory

    The goal of this work is to identify molecular markers associated with the sex chromosomes in rainbow trout to study the mode of sex determination mechanisms in this species. Using the RAPD assay and bulked segregant analysis, two markers were identified that generated polymorphi...

  15. Spot counting on fluorescence in situ hybridization in suspension images using Gaussian mixture model

    NASA Astrophysics Data System (ADS)

    Liu, Sijia; Sa, Ruhan; Maguire, Orla; Minderman, Hans; Chaudhary, Vipin

    2015-03-01

    Cytogenetic abnormalities are important diagnostic and prognostic criteria for acute myeloid leukemia (AML). A flow cytometry-based imaging approach for FISH in suspension (FISH-IS) was established that enables the automated analysis of several log-magnitude higher number of cells compared to the microscopy-based approaches. The rotational positioning can occur leading to discordance between spot count. As a solution of counting error from overlapping spots, in this study, a Gaussian Mixture Model based classification method is proposed. The Akaike information criterion (AIC) and Bayesian information criterion (BIC) of GMM are used as global image features of this classification method. Via Random Forest classifier, the result shows that the proposed method is able to detect closely overlapping spots which cannot be separated by existing image segmentation based spot detection methods. The experiment results show that by the proposed method we can obtain a significant improvement in spot counting accuracy.

  16. Electron Paramagnetic Resonance and Fluorescence In Situ Hybridization-Based Investigations of Individual Doses for Persons Living at Metlino in the Upper Reaches of the Techa River

    SciTech Connect

    Degteva, M. O.; Anspaugh, L. R.; Akleyev, A V.; Jacob, Peter; Ivanov, Denis V.; Wieser, Albrecht; Vorobiova, M I.; Shishkina, Elena A.; Shved, Valentina A.; Vozilova, Alexandra; Bayankin, Sergey N.; Napier, Bruce A.

    2005-02-01

    Waterborne releases to the Techa River from the Mayak Production Association in Russia during 1949-1956 resulted in significant doses to persons living downstream; the most contaminated village was Metlino, about 7 km from the site of release. Internal and external doses have been estimated for these residents using the Techa River Dosimetry System-2000 (TRDS-2000); the primary purpose is to support epidemiological studies of the members of the Extended Techa River Cohort. Efforts to validate the calculations of external and internal dose are considered essential. One validation study of the TRDS-2000 system has been performed by the comparison of calculated doses to quartz from bricks in old buildings at Metlino with those measured by luminescence dosimetry. Two additional methods of validation considered here are electron paramagnetic resonance (EPR) measurements of teeth and fluorescence in situ hybridization (FISH) measurements of chromosome translocations in circulating lymphocytes. For electron paramagnetic resonance, 36 measurements on 26 teeth from 16 donors from Metlino were made at the GSF-National Research Center for Environment and Health (16 measurements) and the Institute of Metal Physics (20 measurements); the correlation among measurements made at the two laboratories has been found to be 0.99. Background measurements were also made on 218 teeth (63 molars, 128 premolars, and 27 incisors). Fluorescence in situ hybridization measurements were made for 31 residents of Metlino. These measurements were handicapped by the analysis of a limited number of cells; for several individuals no stable translocations were observed. Fluorescence in situ hybridization measurements were also made for 39 individuals believed to be unexposed. The EPR- and FISH-based estimates agreed well for permanent residents of Metlino: 0.67 +/- 0.21 Gy and 0.48 +/- 0.18 Gy (mean +/- standard error of the mean), respectively. Results of the two experimental methods also agreed well with the estimates derived from the use of the TRDS-2000. For all persons investigated according to each technique, the EPR-measured dose to enamel was 0.55 +/- 0.17 Gy, and the TRDS-2000 prediction for the dose to enamel for these individuals is 0.55 +/- 0.07 Gy. The fluorescence in situ hybridization-based dose, 0.38 +/- 0.10 Gy, compared well to the TRDS-2000 prediction of external dose, 0.31 +/- 0.03 Gy, to red bone marrow for these persons. Validation of external doses at the remaining villages is an active area of investigation.

  17. Novel karyotype in the Ullrich-Turner syndrome - 45,X/46,X,r(X)/46,X,dic(X) - investigated with fluorescence in situ hybridization

    SciTech Connect

    Robson, L.; Jackson, J.; Cowell, C.; Sillence, D.; Smith, A.

    1994-04-15

    A 10-year-old girl with Ullrich-Turner syndrome was found to have the novel karyotype 45,X/46,X,r(X)(p11q11)/46,X,dic(X)(p11). Fluorescence in situ hybridization (FISH) with the {alpha} satellite X centromere probe established the origin of the small ring chromosome. Scanning a large number of cells by interphase FISH showed that the dicentric (X) was the least prevalent cell line. The common breakpoint of Xp11 suggests a sequence of errors as the mechanism whereby these 3 distinct cell lines have arisen. 11 refs., 4 figs., 1 tab.

  18. Cytogenetic, spectral karyotyping, fluorescence in situ hybridization, and comparative genomic hybridization characterization of two new secondary leukemia cell lines with 5q deletions, and MYC and MLL amplification.

    PubMed

    Knutsen, Turid; Pack, Svetlana; Petropavlovskaja, Maria; Padilla-Nash, Hesed; Knight, Clement; Mickley, Lyn A; Ried, Thomas; Elwood, Patrick C; Roberts, Susan J

    2003-07-01

    Cytogenetic studies of patients with therapy-induced acute myeloid leukemia (t-AML) have demonstrated whole chromosome loss or q-arm deletion of chromosomes 5 and/or 7 in a majority of cases. We have established two cell lines, SAML-1 and SAML-2, from two patients who developed t-AML after radiation and chemotherapy for Hodgkin disease. In both cases, the leukemia cells contained 5q deletions. SAML-1 has 58 chromosomes and numerous abnormalities, including der(1)(1qter-->1p22::5q31-->5qter), der(5)(5pter-->5q22::1p22-->1pter), +8, der(13)i(13)(q10)del(13)(q11q14.1), and t(10;11). Fluorescence in situ hybridization (FISH) with unique sequence probes for the 5q31 region showed loss of IL4, IL5, IRF1, and IL3, and translocation of IL9, DS5S89, EGR1, and CSFIR to 1p. SAML-2 has 45 chromosomes, del(5)(q11.2q31) with a t(12;13)ins(12;5), leading to the proximity of IRF1 and RB1, and complex translocations of chromosomes 8 and 11, resulting in amplification of MYC and MLL. Comparative genomic hybridization and spectral karyotyping were consistent with the G-banding karyotype and FISH analyses. Because a potential tumor suppressor(s) in the 5q31 region has yet to be identified, these cell lines should prove useful in the study of the mechanisms leading to the development of t-AML. PMID:12759925

  19. Fluorescence in situ hybridization-based karyotyping of soybean translocation lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean [Glycine max (L.) Merr.] is a major crop species and a target of a substantial investment of genomic and genetic studies; yet, in contrast to other plant species, relatively few chromosomal aberrations have been identified and characterized in soybean. This is due in part to the difficulty ...

  20. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...assays as an aid in the detection, counting and classification of cells based on recognition of cellular color, size, and shape, and in the detection and enumeration of FISH signals in interphase nuclei of formalin-fixed, paraffin-embedded human tissue...

  1. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...assays as an aid in the detection, counting and classification of cells based on recognition of cellular color, size, and shape, and in the detection and enumeration of FISH signals in interphase nuclei of formalin-fixed, paraffin-embedded human tissue...

  2. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...assays as an aid in the detection, counting and classification of cells based on recognition of cellular color, size, and shape, and in the detection and enumeration of FISH signals in interphase nuclei of formalin-fixed, paraffin-embedded human tissue...

  3. QUANTITATIVE FLUORESCENCE IN SITU HYBRIDIZATION OF AUREOBASIDIUM PULLULANS ON MICROSCOPE SLIDES AND LEAF SURFACES. (R823845)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  4. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos

    E-print Network

    Ji, Ni

    In C. elegans, the expression pattern of a gene provides important clues to understanding its biological function. To accurately depict endogenous transcriptional activity, a highly sensitive method is required to measure ...

  5. Rapid method for measuring clastogenic fingerprints using fluorescence in situ hybridization

    DOEpatents

    Lucas, Joe N. (San Ramon, CA)

    2000-01-01

    A method is provided for determining a clastogenic signature of a sample of chromosomes by quantifying a frequency of a first type of chromosome aberration present in the sample; quantifying a frequency of a second, different type of chromosome aberration present in the sample; and comparing the frequency of the first type of chromosome aberration to the frequency of the second type of chromosome aberration. A method is also provided for using that clastogenic signature to identify a clastogenic agent or dosage to which the cells were exposed.

  6. Fluorescence in situ hybridization and spectral imaging analysisof human oocytes and first polar bodies

    SciTech Connect

    Weier, Heinz-Ulli G.; Weier, Jingly F.; Oter Renom, Maria; Zheng,Xuezhong; Colls, Pere; Nureddin, Aida; Pham, Chau D.; Chu, Lisa W.; Racowsky, Catherine; Munne, Santiago

    2004-10-06

    We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18 or 21 in failed-fertilized human oocytes.While abnormalities involving chromosome 16 showed an age-dependant increase, results for the other chromosomes did not show statistically significant differences between the three age groups <35 yrs, 35-39 yrs, and >39 yrs. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Thus, for a pilot study investigating a more comprehensive analysis of oocytes and their corresponding first polar bodies (1PBs), we developed a novel 8-probe chromosome enumeration scheme using FISH and SIm.

  7. Interphase study by fluorescence in situ hybridization of spermatozoa of a paracentric inversion heterozygote

    SciTech Connect

    Brock, J.K.; Best, R.G.

    1994-09-01

    Cytogenetic studies of peripheral lymphocytes were initiated on a couple with a history of three spontaneous 1st trimester losses and oligospermia. The results revealed the woman to have a normal female karyotype. The man had a karyotype of 46,XY,inv(2)(q14.2q24.3). Interphase sperm studies were offered as an attempt to quantify the relative proportion of sperm with acentric and dicentric chromosome No. 2 in response to the couple`s concern that chromosomally unbalanced sperm resulting from recombination of the inversion was the primary cause of pregnancy losses. Slides were prepared and processed as previously described. A two-color probe cocktail consisting of a biotinylated {alpha}-satellite probe for No. 2 and a digoxigenin-labeled probe for {alpha}-satellite No. 17 as an internal control was employed. Among 496 cells with a single signal for chromosome No. 17, we observed 2 with no signal for chromsome No. 2, 492 with a single signal for No. 2, and 2 cells with 2 signals for No. 2. These findings indicate that the proportion of unbalanced recombinant sperm is probably under 1%, and that other factors are likely to be involved in the etiology of this couple`s pregnancy losses.

  8. Partial trisomy 11q involving chromosome 1 detected by fluorescence in situ hybridization

    SciTech Connect

    McCorquodale, M.; Bereziouk, O.; McCorquodale, D.J.

    1994-09-01

    Partial trisomy 11q was detected in an infant delivered 3-4 weeks prematurely. The phenotype included slanted palpebral fissures, high arched palate, developmental delay, microcephaly, and cardiac defects, all of which occur in the majority of cases with this syndrome. Other features included a column-shaped skull, preauricular pit, single palmar crease, short, broad great toes, flat occiput, unilateral kidney agenesis, and strabismus. Chromosomes obtained from peripheral blood cells revealed the presence of extra material on the long arm of chromosome 1. The G-banding pattern of this extra material indicated that it might be derived from chromosome 1 or 11. Chromosomal {open_quotes}paints{close_quotes} showed that it was not chromosome 1 material, but was chromosome 11 material extending from band q21 to qter. Partial trisomy 11q arising from translocation of the 11q material to chromosome 2, 3, 4, 5, 6, 9, 10, 13, 17, 21, 22, and X has been reported previously, whereas translocation to chromosome 1 has not. The chromosome to which the 11q material is translocated does not alter the most frequent features of the partial trisomy 11q syndrome, but may influence other less common features.

  9. Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization.

    PubMed

    Bisht, M S; Mukai, Y

    2000-12-01

    Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species. PMID:11280009

  10. Fluorescence in situ hybridization for detection of classical propionibacteria with specific 16S rRNA-targeted probes and its application to enumeration in Gruyère cheese.

    PubMed

    Babot, Jaime D; Hidalgo, Maximiliano; Argañaraz-Martínez, Eloy; Apella, María C; Perez Chaia, Adriana

    2011-01-31

    The classical or dairy propionibacteria have well-documented industrial applications and have been proposed for probiotic applications. Given their industrial importance it is necessary to employ fast and reliable techniques to monitor the growth during products elaboration, industrial fermentations or the intestinal transit. Therefore, the aim of this investigation was to design oligonucleotide probes targeting the 16S rRNA of dairy propionibacteria and optimise the fluorescence in situ hybridization (FISH) protocol to detect these bacteria. Two specific probes were in silico designed to detect Propionibacterium freudenreichii and P. jensenii, named Pfr435 and Pj446 respectively. The FISH protocol was optimised for the hybridisation of propionibacteria cells with the universal probe Eub338 and the designed probes. These probes were assayed in situ for their specificity to hybridise species of propionibacteria by observation using fluorescence microscopy and results were compared with the probe Pap446 previously designed for P. acidipropionici. Probes Pap446, Pfr435 and Pj446 were also evaluated by fluorescence spectrophotometry to assess the influence of cells physiological state during growth in batch culture in the fluorescence intensity. The maximum fluorescence intensity was observed at the onset of the stationary phase of growth and was then reduced. However, changes on the cells permeability did not reduce the efficiency of 16S rRNA hybridisation with the fluorescence-labelled probes. Propionibacteria counts obtained by FISH and plate count methods were compared in a commercial Gruyère cheese. The results showed that this method can be used as a rapid technique for the enumeration of these bacteria in cheese samples. PMID:21276635

  11. Intratumoral Patterns of Clonal Evolution in Meningiomas as Defined by Multicolor Interphase Fluorescence in Situ Hybridization (FISH)

    PubMed Central

    Sayagués, José María; Tabernero, María Dolores; Maíllo, Angel; Espinosa, Ana; Rasillo, Ana; Díaz, Pedro; Ciudad, Juana; López, Antonio; Merino, Marta; Gonçalves, Jesús María; Santos-Briz, Angel; Morales, Francisco; Orfao, Alberto

    2004-01-01

    Meningiomas are cytogenetically heterogeneous tumors in which chromosome gains and losses frequently occur. Based on the intertumoral cytogenetic heterogeneity of meningiomas, hypothetical models of clonal evolution have been proposed in these tumors which have never been confirmed at the intratumoral cell level. The aim of this study was to establish the intratumoral patterns of clonal evolution associated with chromosomal instability in individual patients as a way to establish tumor progression pathways in meningiomas and their relationship with tumor histopathology and behavior. A total of 125 meningioma patients were analyzed at diagnosis. In all cases, multicolor interphase fluorescence in situ hybridization (iFISH) studies were performed on fresh tumor samples for the detection of quantitative abnormalities for 11 different chromosomes. In addition, overall tumor cell DNA content was measured in parallel by flow cytometry. iFISH studies were also performed in parallel on tissue sections in a subset of 30 patients. FISH studies showed that 56 (45%) of the 125 cases analyzed had a single tumor cell clone, all these cases corresponding to histologically benign grade I tumors. In the remaining cases (55%) more than one tumor cell clone was identified: two in 45 cases (36%), three in 19 (15%), and four or more clones in five cases (4%). Overall, flow cytometric analysis of cell DNA contents showed the presence of DNA aneuploidy in 44 of these cases (35%), 30% corresponding to DNA hyperdiploid and 5% to hypodiploid cases; from the DNA aneuploid cases, 35 (28%) showed two clones and 9 (7%) had three or more clones. A high degree of correlation (r ? 0.89; P < 0.001) was found between FISH and flow cytometry as regards the overall quantitative DNA changes detected with both techniques, the former being more sensitive. Among the cases with chromosome abnormalities, the earliest tumor cell clone observed was frequently characterized by the loss of one or more chromosomes (64% of all meningiomas); loss of either a single chromosome 22 or, less frequently, of a sex chromosome (X or Y) and del (1p) was commonly found as the single initial cytogenetic aberration (30%, 5%, and 5% of the cases, respectively). Interestingly, an isolated loss of chromosome 22 was only found as the initial abnormality in one out of 14 atypical/anaplastic meningiomas, while the same cytogenetic pattern was present in the ancestral tumor cell clone of 32% of the benign tumors. Cytogenetic patterns based on chromosome gains were found in the ancestral tumor cell clone in 4% of the patients, 2% corresponding to tetraploid tumors. Overall, cytogenetic evolution of the earliest tumor cell clones was frequently associated with tetraploidization (31%). Our results show that meningiomas are genetically heterogeneous tumors that display different patterns of numerical chromosome changes, with the presence of more than one tumor cell clone detected in almost half of the cases including all atypical/anaplastic cases. Interestingly, the pathways of intratumoral clonal evolution observed in the benign tumors were different from those observed in atypical/anaplastic meningiomas, suggesting that the latter tumors might not always represent a more advanced stage of histologically benign meningiomas. PMID:15507670

  12. In Situ Measurement of Bioluminescence and Fluorescence in an Integrated

    E-print Network

    Sinskey, Anthony J.

    In Situ Measurement of Bioluminescence and Fluorescence in an Integrated Microbioreactor Andrea of bioluminescence and fluorescence from bacterial cultures grown in 50 mL instrumented microbioreactors. Results measurements; bioluminescence; fluorescence; instrumented microbioreactors; Escheri- chia coli; luxCDABE; gfp

  13. Multicolor fluorescence in situ hybridization with centromeric DNA probes as a new approach to distinguish chromosome breakage from aneuploidy in interphase cells and micronuclei

    SciTech Connect

    Eastmond, D.A.; Rupa, D.S.; Chen, H.W.; Hasegawa, L.

    1993-12-31

    Chromosomal abnormalities are believed to contribute significantly to human reproductive failure, carcinogenesis and other pathophysiological conditions. For example, approximately 15% of recognized pregnancies terminate in spontaneous abortion, and of these approximately 30% have been shown to be chromosomally abnormal. The contribution of chromosomal abnormalities to early embryonic and fetal death appears to decrease with gestational age, suggesting that as many as 67% of the aborted embryos in early embryonic deaths are chromosomally abnormal. Furthermore, clinically significant chromosomal abnormalities can also be found to be present in approximately 0.58 to 0.67% of live births. These figures indicate that within a given year, hundreds of thousands of chromosomally abnormal babies will be born throughout the world and additional millions of chromosomally abnormal embryos will have been spontaneously aborted. For the past several years, our research has focused on utilizing new molecular cytogenetic techniques to develop assays for detecting aneuploidy-inducing agents in mammalian cells. One approach that we have sucessfully employed involves the use of fluorescence in situ hybridization with chromosome-specific DNA probes to determine the number of copies of a representative chromosome present within the nucleus following chemical exposure. DNA sequences (probes) which hybridize to blocks of repetitive centromeric DNA on specific chromosomes have been developed for most of the human chromosomes. In situ hybridization with these probes results in the staining of a compact chromosomal region which can be easily detected in interphase nuclei. The presence of 3 (or more) hybridization domains in an interphase nucleus indicates the presence of three centromeric regions and has been presumed to indicate that three copies of the entire chromosome were present in the nucleus.

  14. Non-Hodgkin Lymphoma (NHL) subtypes defined by common translocations: utility of fluorescence in situ hybridization (FISH) in a case-control study

    PubMed Central

    Chang, Cindy M.; Schroeder, Jane C.; Huang, Wen-Yi; Dunphy, Cherie H.; Baric, Ralph S.; Olshan, Andrew F.; Dorsey, Kathleen C.; Dent, Georgette A.; Cerhan, James R.; Lynch, Charles F.; Rothman, Nathaniel; Cantor, Kenneth P.; Blair, Aaron

    2009-01-01

    We used fluorescence in situ hybridization (FISH) assays to identify t(14;18) translocations in archival paraffin-embedded tumor sections from non-Hodgkin lymphoma (NHL) cases enrolled in a population-based study. t(14;18) was identified in 54% of 152 cases, including 39% of diffuse large cell lymphomas (26 of 66 cases) and 84% of follicular lymphomas (36 of 43 cases). Eighty-seven percent of t(14;18)-positive cases and 57% of t(14;18)-negative cases expressed bcl-2. FISH assays detected twice as many t(14;18)-positive follicular lymphomas as PCR assays. Overall, study findings support the use of FISH assays to detect t(14;18) in archival tumor samples for epidemiologic studies of NHL subtypes. PMID:19505720

  15. Dicentric chromosome aberration analysis using giemsa and centromere specific fluorescence in-situ hybridization for biological dosimetry: An inter- and intra-laboratory comparison in Indian laboratories.

    PubMed

    Bhavani, M; Tamizh Selvan, G; Kaur, Harpreet; Adhikari, J S; Vijayalakshmi, J; Venkatachalam, P; Chaudhury, N K

    2014-09-01

    To facilitate efficient handling of large samples, an attempt towards networking of laboratories in India for biological dosimetry was carried out. Human peripheral blood samples were exposed to (60)Co ?-radiation for ten different doses (0-5Gy) at a dose rate of 0.7 and 2Gy/min. The chromosomal aberrations (CA) were scored in Giemsa-stained and fluorescence in-situ hybridization with centromere-specific probes. No significant difference (p>0.05) was observed in the CA yield for given doses except 4 and 5Gy, between the laboratories, among the scorers and also staining methods adapted suggest the reliability and validates the inter-lab comparisons exercise for triage applications. PMID:25014548

  16. Identification of the origin of marker chromosomes by two-color fluorescence in situ hybridization and polymerase chain reaction in azoospermic patients.

    PubMed

    Wei, C L; Cheng, J L; Yang, W C; Li, L Y; Cheng, H C; Fu, J J

    2015-01-01

    Y chromosomal microdeletions at the azoospermia factor locus and chromosome abnormalities have been implicated as the major causes of idiopathic male infertility. A marker chromosome is a structurally abnormal chromosome in which no part can be identified by cytogenetics. In this study, to identify the origin of the marker chromosomes and to perform a genetic diagnosis of patients with azoospermia, two-color fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) techniques were carried out. The marker chromosomes for the two patients with azoospermia originated in the Y chromosome; it was ascertained that the karyotype of both patients was 46,X, ish del(Y)(q11)(DYZ3+, DXZ1-). The combination of two-color FISH and PCR techniques is an important method for the identification of the origin of marker chromosomes. Thus, genetic counseling and a clear genetic diagnosis of patients with azoospermia before intracytoplasmic sperm injection or other clinical managements are important. PMID:26600507

  17. Comparative mapping of DNA probes derived from the V{sub k} immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization

    SciTech Connect

    Arnold, N.; Wienberg, J.; Ermert, K.

    1995-03-01

    Fluorescence in situ hybridization (FISH) of cosmid clones of human V{sub K} gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the K locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo Pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore, be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V{sub K}I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V{sub K} region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution. 18 refs., 2 figs.

  18. Clinical and cytogenetic findings in seven cases of inverted duplication of 8p with evidence of a telomeric deletion using fluorescence in situ hybridization

    SciTech Connect

    Guo, Wen-Jun; Callif-Daley, F.; Zapata, M.C.; Miller, M.E.

    1995-09-11

    We report on the clinical and cytogenetic findings in 7 cases of inverted duplication of region 8p11.2-p23. The phenotype of inv dup (8p) compiled from this series and the literature (N = 29) consists of severe mental retardation (100%), minor facial alterations (97%), agenesis of the corpus callosum (80%), hypotonia (66%), orthopedic abnormalities (58%), scoliosis/kyphosis (40%), and congenital heart defect (26%). A telomeric deletion of region 8p23.3-pter was confirmed in 3 of our cases studied using fluorescent in situ hybridization with a telomeric probe for 8p. Thus, these karyotypes are inv dup del(8) (qter{r_arrow} p23.1::p23.1{r_arrow}p11.2:). Our findings suggest that most cases of inv dup(8p) probably have a telomeric deletion. 20 refs., 4 figs., 2 tabs.

  19. Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

    1998-01-01

    A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

  20. Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization? †

    PubMed Central

    Smolina, Irina; Lee, Charles; Frank-Kamenetskii, Maxim

    2007-01-01

    An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes. PMID:17293504

  1. Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes

    PubMed Central

    Wullings, B. A.; Van Beuningen, A. R.; Janse, J. D.; Akkermans, A. D. L.

    1998-01-01

    During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas. PMID:9797321

  2. Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

    PubMed Central

    Zoetendal, Erwin G.; Ben-Amor, Kaouther; Harmsen, Hermie J. M.; Schut, Frits; Akkermans, Antoon D. L.; de Vos, Willem M.

    2002-01-01

    A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract. PMID:12200269

  3. Impact of updated HER2 testing guidelines in breast cancer-re-evaluation of HERA trial fluorescence in situ hybridization data.

    PubMed

    Stoss, Oliver C; Scheel, Andreas; Nagelmeier, Iris; Schildhaus, Hans-Ulrich; Henkel, Thomas; Viale, Giuseppe; Jasani, Bharat; Untch, Michael; Rüschoff, Josef

    2015-12-01

    Recently the American Society of Clinical Oncology and the College of American Pathologists have updated their clinical practice guidelines for HER2 testing in breast cancer. In order to evaluate these new recommendations, we have re-assessed the HER2 status of 6018 breast cancer cases of the screening population for the HERceptin adjuvant (HERA) trial that were originally centrally tested by fluorescence in situ hybridization based on the FDA-released test guidelines. According to the most recent 2013 ASCO/CAP recommendations, 3380 (56.2%) cases were classified as HER2 positive compared with 3359 (55.8%) applying the HERA/FDA scheme and 3339 (55.5%) applying the 2007 ASCO/CAP guidelines. Twenty-one cases switched from negative (HERA/FDA scheme) to positive (2013 ASCO/CAP guidelines). This group is characterized by a mean HER2 gene copy number of ?6.0, polysomy or co-amplification of CEP17 with an average CEP17 count of 5, and with HER2 receptor overexpression in 75% of cases. On the basis of the HER2 gene copy number alone, we observe 494 cases (8.2%) that are in the equivocal range. Most of these cases (>80%) were also nondecisive by immunohistochemistry (score 2+) irrespective of whether ratio was <2.0>. The number of equivocal cases that would require HER2 reflex testing decreases to 113 (1.9%) if in addition to the HER2 gene copy number also the ratio of HER2 and CEP17 copy numbers is considered via dual-color in situ hybridization. The combination of applying the HER2 mean gene copy number as well as the HER2/CEP17 ratio to define equivocal test decisions by fluorescence in situ hybridization as proposed by the current ASCO/CAP guidelines appears to be a more optimum approach to adopt in order to avoid or minimize reporting of false negative results. Using the mean HER2 gene copy number alone for decision making results in a significant increase of equivocal cases. PMID:26403781

  4. Limited utility of fluorescence in situ hybridization for common abnormalities of myelodysplastic syndrome at first presentation and follow-up of myeloid neoplasms.

    PubMed

    Seegmiller, Adam C; Wasserman, Allison; Kim, Annette S; Kressin, Megan K; Marx, Edward R; Zutter, Mary M; Mosse, Claudio A

    2014-03-01

    Fluorescence in situ hybridization for abnormalities common to myelodysplastic syndrome (MDS FISH) is often used with traditional karyotype in the diagnosis and monitoring of myeloid neoplasms. However, its value in these roles has been questioned. To evaluate its utility, we compared MDS FISH results with karyotype in 544 bone marrow specimens obtained for diagnosis (180 cases) or follow-up (364 cases) of myeloid neoplasia. We found excellent concordance between FISH and karyotype, such that FISH is rarely abnormal (1.7% at diagnosis and 3.0% at follow-up) in cases with normal karyotype. Even in the rare discordant cases, the abnormal FISH has little or no clinical value. Thus, we propose that this test should be limited to cases with inadequate karyotype only. Such guidelines could result in significant cost savings with no impact on patient diagnosis. PMID:23876099

  5. Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera).

    PubMed

    Yue, Dominique; Nordhoff, Marcel; Wieler, Lothar H; Genersch, Elke

    2008-06-01

    American foulbrood (AFB) is a bacterial disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae. Although AFB and its aetiological agent are described now for more than a century, the general and molecular pathogenesis of this notifiable disease is poorly understood. We used fluorescence in situ hybridization (FISH) performed with P. larvae-specific, 16S rRNA-targeted oligonucleotide probes to analyse the early steps in the pathogenesis of American foulbrood. The following chain of events could be demonstrated: (i) the spores germinate in the midgut lumen, (ii) the vegetative bacteria massively proliferate within the midgut before, and (iii) they start to locally breach the epithelium and invade the haemocoel. The paracellular route was shown to be the main mechanism for invasion contrasting earlier hypotheses of phagocytosis of P. larvae. Invasion coincided with the death of the host implicating that the penetration of the midgut epithelium is a critical step determining the time of death. PMID:18331334

  6. Mosaic vs. nonmosaic trisomy 9: Report of a liveborn infant evaluated by fluorescence in situ hybridization and review of the literature

    SciTech Connect

    Cantu, E.S.; Eicher, D.J.; Shashidhar Pai, G.; Donahue, C.J.; Harley, R.A.

    1996-04-24

    We report on a newborn infant with multiple congenital anomalies and apparent nonmosaic trisomy 9 in the blood (by conventional cytogenetic studies) who died shortly after birth. Clinical observations at birth and autopsy are compared with phenotypes of mosaic and nonmosaic trisomy 9 cases reported previously. Unlike the initial cytogenetic analysis, fluorescence in situ hybridization (FISH) studies of metaphase and interphase blood cells and skin fibroblasts detected the presence of euploid and trisomy 9 cells. These results suggest that earlier reports of trisomy 9, which relied on conventional chromosome analysis of a few metaphase cells and/or only one tissue type, may not have excluded mosaicism, and that trisomy 9 may be viable only in the mosaic state. 39 refs., 3 figs., 2 tabs.

  7. Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH) †,‡

    PubMed Central

    Packard, Michelle M.; Shusteff, Maxim; Alocilja, Evangelyn C.

    2012-01-01

    Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation. PMID:25586031

  8. Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: a comprehensive survey and analysis.

    PubMed

    Chung, Chen-yo; Cook, Charles E; Lin, Gee-way; Huang, Ting-Yu; Chang, Chun-che

    2014-06-01

    RNA in situ hybridization (ISH), including chromogenic ISH (CISH) and fluorescent ISH (FISH), has become a powerful tool for revealing the spatial distribution of gene transcripts in model organisms. Previously, we developed a robust protocol for whole-mount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomic model. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow, using confocal microscopy, clearer visualization of target messenger RNAs (mRNAs) - including those subcellularly localized and those with spatially overlapping expression. We find that Fast dye-based substrate fluorescence (SF), tyramide signal amplification (TSA), and TSA Plus all enable identifying gene expression thanks to multiplex amplification of fluorescent signals. By contrast, methods of direct fluorescence (DF) do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plus for most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvas1 and Appiwi2 in the germ cells. For detection of multiple gene targets using double FISH, we recommend: (i) TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ cells, as proteinase K treatment can be omitted; and (ii) SF/TSA Plus for other gene targets such as Apen1 and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due to signal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea aphid. We anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from which useful insights into development and evolution may be obtained. PMID:24850784

  9. Fluorescent in situ hybridization shows DIPLOSPOROUS located on one of the NOR chromosomes in apomictic dandelions (Taraxacum) in the absence of a large hemizygous chromosomal region.

    PubMed

    Vašut, Radim J; Vijverberg, Kitty; van Dijk, Peter J; de Jong, Hans

    2014-11-01

    Apomixis in dandelions (Taraxacum: Asteraceae) is encoded by two unlinked dominant loci and a third yet undefined genetic factor: diplosporous omission of meiosis (DIPLOSPOROUS, DIP), parthenogenetic embryo development (PARTHENOGENESIS, PAR), and autonomous endosperm formation, respectively. In this study, we determined the chromosomal position of the DIP locus in Taraxacum by using fluorescent in situ hybridization (FISH) with bacterial artificial chromosomes (BACs) that genetically map within 1.2-0.2 cM of DIP. The BACs showed dispersed fluorescent signals, except for S4-BAC 83 that displayed strong unique signals as well. Under stringent blocking of repeats by C0t-DNA fragments, only a few fluorescent foci restricted to defined chromosome regions remained, including one on the nucleolus organizer region (NOR) chromosomes that contains the 45S rDNAs. FISH with S4-BAC 83 alone and optimal blocking showed discrete foci in the middle of the long arm of one of the NOR chromosomes only in triploid and tetraploid diplosporous dandelions, while signals in sexual diploids were lacking. This agrees with the genetic model of a single dose, dominant DIP allele, absent in sexuals. The length of the DIP region is estimated to cover a region of 1-10 Mb. FISH in various accessions of Taraxacum and the apomictic sister species Chondrilla juncea, confirmed the chromosomal position of DIP within Taraxacum but not outside the genus. Our results endorse that, compared to other model apomictic species, expressing either diplospory or apospory, the genome of Taraxacum shows a more similar and less diverged chromosome structure at the DIP locus. The different levels of allele sequence divergence at apomeiosis loci may reflect different terms of asexual reproduction. The association of apomeiosis loci with repetitiveness, dispersed repeats, and retrotransposons commonly observed in apomictic species may imply a functional role of these shared features in apomictic reproduction, as is discussed. PMID:25760668

  10. Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

    PubMed

    Horn, Heike; Bausinger, Julia; Staiger, Annette M; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M Michaela; Rosenwald, Andreas; Ott, German

    2014-01-01

    Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches. PMID:24733537

  11. Whole-mount in situ hybridization using DIG-labeled probes in planarian.

    PubMed

    Rybak-Wolf, Agnieszka; Solana, Jordi

    2014-01-01

    In recent years freshwater flatworms (planarian) have become a powerful model for studies of regeneration and stem cell biology. Whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) are key and most commonly used techniques to determine and visualize gene expression patterns in planaria. Here, we present the established version of whole-mount in situ hybridization (WISH) and whole-mount fluorescence in situ hybridization (WFISH) protocol optimized over the last years by several labs from the rapidly growing planaria field and give an overview of recently introduced modifications which can be critical in the study of low abundant transcripts. PMID:25218375

  12. Re-appraisal of the phylogeny and fluorescence in situ hybridization probes for the analysis of the Competibacteraceae in wastewater treatment systems.

    PubMed

    McIlroy, Simon J; Nittami, Tadashi; Kanai, Eri; Fukuda, Junji; Saunders, Aaron M; Nielsen, Per Halkjaer

    2015-04-01

    Members of the family Competibacteraceae are common in wastewater treatment plants (WWTPs) designed for enhanced biological phosphorus removal (EBPR) and are putatively deleterious to the process of P removal. Their ability to accumulate large amounts of polyhydroxyalkanoates is also suggested to be of potential commercial interest for bioplastic production. In this study we have updated the 16S rRNA-based phylogeny of the Competibacter and the Plasticicumulans lineages. The former is delineated by 13 clades including two described genera; 'Ca.?Competibacter' and 'Ca.?Contendobacter'. The oligonucleotide probes used for detection of the family by fluorescence in situ hybridization (FISH) were re-evaluated and designed for coverage of these clades. Surveys of full-scale WWTPs based on 16S rRNA gene amplicon sequencing and FISH analysis indicate that a number of member clades always coexist, with their relative abundances varying substantially between and temporally within plants. The hypothesis that these differences are based on niche partitioning is supported by marked phenotypic differences between clades. An in-depth understanding of the ecology of the family requires further studies of the metabolism of individual clades in situ. The proposed phylogeny and FISH probes will provide the foundation for such studies. PMID:25224028

  13. Mapping of low-frequency chimeric yeast artificial chromosome libraries from human chromosomes 16 and 21 by fluorescence in situ hybridization and quantitative image analysis

    SciTech Connect

    Marrone, B.L.; Campbell, E.W.; Anzick, S.L.; Shera, K.; Campbell, M.; Yoshida, T.M.; McCormick, M.K.; Deaven, L. )

    1994-05-01

    Yeast artificial chromosome (YAC) clones from low-frequency chimeric libraries of human chromosomes 16 and 21 were mapped onto human diploid fibroblast metaphase chromosomes using fluorescence in situ hybridization (FISH) and digital imaging microscopy. YACs mapped onto chromosome 21 were selected to provide subregional location and ordering of known and unknown markers on the long arm of chromosome 21, particularly in the Down syndrome region (q22). YACs mapped onto chromosome 16 were selected to overlap regions spanning chromosome 16 cosmid maps. YAC clones were indirectly labeled with fluorescein, and the total DNA of the chromosome was counterstained with propidium iodide. A single image containing both the FISH signal and the whole chromosome was acquired for each chromosome of interest containing the fluorescent probe signal in a metaphase spread. From the digitized image, the fluorescence intensity profile through the long axis of the chromosome gave the total chromosome length and the probe position. The map position of the probe was expressed as the fractional length (FL) of the total chromosome relative to the end of the short arm (Flpter). From each clone hybridized, 20-40 chromosome images were analyzed. Thirty-eight YACs were mapped onto chromosome 16, and their FLs were distributed along the short and long arms. On chromosome 21, 47 YACs were mapped, including 12 containing known markers. To confirm the order of a dense population of YACs within the Down syndrome region, a two-color mapping strategy was used in which an anonymous YAC was located relative to one or two known markers on the metaphase chromosome. The chromosome FL maps have a 1- to 2-Mb resolution, and the FL measurement of each probe has a typical standard error of 0.5-1 Mb. 14 refs., 3 figs., 3 tabs.

  14. Simultaneous 16S and 18S rRNA fluorescence in situ hybridization (FISH) on LR White sections demonstrated in Vestimentifera (Siboglinidae) tubeworms.

    PubMed

    Schimak, Mario P; Toenshoff, Elena R; Bright, Monika

    2012-02-01

    Traditional morphological identification of invertebrate marine species is limited in early life history stages for many taxa. In this study, we demonstrate, by example of Vestimentiferan tubeworms (Siboglinidae, Polychaeta), that the simultaneous fluorescence in situ hybridization (FISH) of both eukaryotic host and bacterial symbiont cells is possible on a single semi-thin (1 ?m) section. This allows the identification of host specimens to species level as well as offering visualization of bacteria distributed within the host tissue. Previously published 18S rRNA host-specific oligonucleotide probes for Riftia pachyptila, Tevnia jerichonana and a newly designed Oasisia alvinae probe, as well as a 16S rRNA probe targeting symbionts found in all host species, were applied. A number of standard fixation and hybridization parameters were tested and optimized for the best possible signal intensity and cellular resolution. Ethanol conserved samples embedded in LR White low viscosity resin yielded the best results with regard to both signal intensity and resolution. We show that extended storage times of specimens does not affect the quality of signals attained by FISH and use our protocol to identify morphologically unidentifiable tubeworm individuals from a small data set, conforming to previous findings in succession studies of the Siboglinidae family. PMID:21507466

  15. mRNA-targeted Fluorescent in Situ Hybridization (FISH) of Gram-negative Bacteria Without Template Amplification or Tyramide Signal Amplification

    SciTech Connect

    Coleman, James R.; Culley, David E.; Chrisler, William B.; Brockman, Fred J.

    2007-12-01

    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 multicopy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact 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 the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.

  16. Detection of single copy genes by two-pass tyramide signal amplification fluorescence in situ hybridization (Two-Pass TSA-FISH) with single oligonucleotide probes.

    PubMed

    Kawakami, Shuji; Kubota, Kengo; Imachi, Hiroyuki; Yamaguchi, Takashi; Harada, Hideki; Ohashi, Akiyoshi

    2010-01-01

    In situ detection of functional genes is informative for understanding microbial physiology. Most methods of detecting functional genes employ multiple oligonucleotides or polynucleotide probes. However, single oligonucleotide probes are superior in terms of specificity and flexibility in probe design. Here we describe the detection of a single copy functional gene, the methyl coenzyme M reductase gene, in a methanogen by two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a single oligonucleotide probe without pre-amplification of target nucleic acids. Locked-nucleic-acid-incorporated DNA probes were employed to achieve high specificity and affinity. Although problems associated with non-removable nonspecific binding of the antibody could not be overcome completely, single copy gene detection was carried out with single mismatch descriminatable specificity; however, only around 15% of cells were detected. The detection rate increased when a multiple copy gene like rrn in Escherichia coli was targeted, indicating that a certain number of target molecules are necessary to achieve a high detection rate. Although possible applications of this technique to environmental samples remain restricted, the results presented the potential of gene detection by FISH with single oligonucleotide probes. PMID:21576847

  17. Validation of a new catalysed reporter deposition-fluorescence in situ hybridization probe for the accurate quantification of marine Bacteroidetes populations.

    PubMed

    Acinas, Silvia G; Ferrera, Isabel; Sarmento, Hugo; Díez-Vives, Cristina; Forn, Irene; Ruiz-González, Clara; Cornejo-Castillo, Francisco M; Salazar, Guillem; Gasol, Josep M

    2015-10-01

    Catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) is a powerful approach to quantify bacterial taxa. In this study, we compare the performance of the widely used Bacteroidetes?CF319a probe with the new CF968 probe. In silico analyses and tests with isolates demonstrate that CF319a hybridizes with non-Bacteroidetes sequences from the Rhodobacteraceae and Alteromonadaceae families. We test the probes' accuracy in 37 globally distributed marine samples and over two consecutive years at the Blanes Bay Microbial Observatory (NW Mediterranean). We also compared the CARD-FISH data with the Bacteroidetes 16S rRNA gene sequences retrieved from 27 marine metagenomes from the TARA Oceans expedition. We find no significant differences in abundances between both approaches, although CF319a targeted some unspecific sequences and both probes displayed different abundances of specific Bacteroidetes phylotypes. Our results demonstrate that quantitative estimations by using both probes are significantly different in certain oceanographic regions (Mediterranean Sea, Red Sea and Arabian Sea) and that CF968 shows seasonality within marine Bacteroidetes, notably large differences between summer and winter that is overlooked by CF319a. We propose CF968 as an alternative to CF319a for targeting the whole Bacteroidetes phylum since it has better coverage, greater specificity and overall better quantifies marine Bacteroidetes. PMID:24890225

  18. Simultaneous Quantification of Active Carbon- and Nitrogen-Fixing Communities and Estimation of Fixation Rates Using Fluorescence In Situ Hybridization and Flow Cytometry

    PubMed Central

    Shepard, Alicia K.; Raes, Eric J.; Waite, Anya M.; Quigg, Antonietta

    2014-01-01

    Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and 14C/15N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean. PMID:25172848

  19. Gene protein detection platform--a comparison of a new human epidermal growth factor receptor 2 assay with conventional immunohistochemistry and fluorescence in situ hybridization platforms.

    PubMed

    Stålhammar, Gustav; Farrajota, Pedro; Olsson, Ann; Silva, Cristina; Hartman, Johan; Elmberger, Göran

    2015-08-01

    Human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are widely used semiquantitative assays for selecting breast cancer patients for HER2 antibody therapy. However, both techniques have been shown to have disadvantages. Our aim was to test a recent automated technique of combined IHC and brightfield dual in situ hybridization-gene protein detection platform (GPDP)-in breast cancer HER2 protein, gene, and chromosome 17 centromere status evaluations, comparing the results in accordance to the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from both 2007 and 2013. The GPDP technique performance was evaluated on 52 consecutive whole slide invasive breast cancer cases with HER2 IHC 2/3+ scoring results. Applying in turns the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from 2007 and 2013 to both FISH and GPDP DISH assays, the HER2 gene amplification results showed 100% concordance among amplified/nonamplified cases, but there was a shift in 4 cases toward positive from equivocal results and toward equivocal from negative results. This might be related to the emphasis on the average HER2 copy number in the 2013 criteria. HER2 expression by IVD market IHC kit (Pathway®) has a strong correlation with GPDP HER2 protein, including a full concordance for all cases scored as 3+ and a reduction from 2+ to 1+ in 7 cases corresponding to nonamplified cases. Gene protein detection platform HER2 protein "solo" could have spared the need for 7 FISH studies. In addition, the platform offered advantages on interpretation reassurance including selecting areas for counting gene signals paralleled with protein IHC expression, on heterogeneity detection, interpretation time, technical time, and tissue expense. PMID:25921313

  20. Agreement between amoA Gene-Specific Quantitative PCR and Fluorescence In Situ Hybridization in the Measurement of Ammonia-Oxidizing Bacteria in Activated Sludge

    PubMed Central

    Lunn, M.; Davenport, R. J.; Swan, D. L.; Read, L. F.; Brown, M. R.; Morais, C.; Curtis, T. P.

    2014-01-01

    Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase (amoA) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers. PMID:25002435

  1. Numerical aberrations of chromosome 8 detected by conventional cytogenetics and fluorescence in situ hybridization in individuals from northern Brazil with gastric adenocarcinoma.

    PubMed

    Assumpção, Paulo Pimentel; Ishak, Geraldo; Chen, Elizabeth Suchi; Takeno, Sylvia Satomi; Leal, Mariana Ferreira; Guimarães, Adriana Costa; Calcagno, Danielle Queiroz; Khayat, André Salim; Demachki, Samia; Smith, Marília de Arruda Cardoso; Burbano, Rommel Rodríguez

    2006-08-01

    Gastric cancer is the third most frequent type of neoplasia and the second most important cause of cancer-related death in the world. In northern Brazil, the state of Pará shows a high incidence of this disease and the capital ranks among cities with the highest incidence of stomach cancer in the world. To evaluate chromosomal aberrations implicated in gastric carcinogenesis, we analyzed 16 samples of gastric adenocarcinoma by fluorescence in situ hybridization using a chromosome 8 alpha-satellite probe and by direct chromosomal analysis techniques. All lesions were classified as at advanced stages according to the recommendations of the Union Internationale Contre le Cancer (UICC). Trisomy 8 was the main finding of this study, observed in all cases. There was no significant difference between chromosome 8 ploidy and localization, stage, or histological type of adenocarcinoma in our sample. The high incidence of alterations we found in chromosome 8 may be a regional characteristic, related to the high incidence of this neoplasm in Pará state and a strong influence of external factors, such as eating habits. This aberration may comprise a cytogenetic subgroup of this neoplasm. Additional investigations are necessary to confirm the involvement of chromosome 8 and to identify genes in this chromosome related to gastric carcinogenesis. PMID:16875936

  2. Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea x A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes.

    PubMed

    Kamstra, S A; Ramanna, M S; de Jeu, M J; Kuipers, A G; Jacobsen, E

    1999-01-01

    A distant hybrid between two diploid species (2n = 2x = 16), Alstroemeria aurea and A. inodora, was investigated for homoeologous chromosome pairing, crossability with A. inodora and chromosome transmission to its BC1 offspring. Fluorescence in situ hybridization (FISH) with two species-specific probes, A001-I (A. aurea specific) and D32-13 (A. inodora specific), was used to analyse chromosome pairing in the hybrid and the genome constitution of its BC1 progeny plants. High frequencies of associated chromosomes were observed in both genotypes of the F1 hybrid, A1P2-2 and A1P4. In the former, both univalents and bivalents were found at metaphase I, whereas the latter plant also showed tri- and quadrivalents. Based on the hybridization sites of DNA probes on the chromosomes of both parental species, it was established that hybrid A1P4 contains a reciprocal translocation between the short arm of chromosome 1 and the long arm of chromosome 8 of A. inodora. Despite regular homoeologous chromosome pairing in 30% of the pollen mother cells, both hybrids were highly sterile. They were backcrossed reciprocally with one of the parental species, A. inodora. Two days after pollination, embryo rescue was applied and, eventually, six BC1 progeny plants were obtained. Among these, two were aneuploids (2n = 2x + 1 = 17) and four were triploids (2n = 3x = 24). The aneuploid plants had originated when the interspecific hybrid was used as a female parent, indicating that n eggs were functional in the hybrid. In addition, 2n gametes were also functional in the hybrid, resulting in the four triploid BC1 plants. Of these four plants, three had received 2n pollen grains from the hybrid and one a 2n egg. Using FISH, homoeologous crossing over between the chromosomes of the two parental species in the hybrid was clearly detected in all BC1 plants. The relevance of these results for the process of introgression and the origin of n and 2n gametes are discussed. PMID:10087627

  3. Assignment of the human diacylglycerol kinase gene (DAGK) to 12q13.3 using fluorescence in situ hybridization analysis

    SciTech Connect

    Hart, T.C.; Zhou, J.; Champagne, C.

    1994-07-01

    A 1-kb cDNA probe specific for the human DAGK gene was prepared by polymerase chain reaction and used to assign it to human chromosome 12 using a human x hamster somatic cell hybrid mapping panel. To determine the chromosomal sublocalization of DAGK, used the 1-kg DAGK cDNA probe to isolate a DAGK-specific phage clone from a human chromosome 12 library by Southern blot techniques. This clone (phEDCDAGK) contained a 17-kb human genomic insert in the phage Charon 40 that hybridized to the 1-kb DAGK cDNA previously characterized.

  4. Radiation-induced chromosome aberrations in ataxia telangiectasia cells: high frequency of deletions and misrejoining detected by fluorescence in situ hybridization

    NASA Technical Reports Server (NTRS)

    Kawata, Tetsuya; Ito, Hisao; George, Kerry; Wu, Honglu; Uno, Takashi; Isobe, Kouichi; Cucinotta, Francis A.

    2003-01-01

    The mechanisms underlying the hyper-radiosensitivity of AT cells were investigated by analyzing chromosome aberrations in the G(2) and M phases of the cell cycle using a combination of chemically induced premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with chromosome painting probes. Confluent cultures of normal fibroblast cells (AG1522) and fibroblast cells derived from an individual with AT (GM02052) were exposed to gamma rays and allowed to repair at 37 degrees C for 24 h. At doses that resulted in 10% survival, GM02052 cells were approximately five times more sensitive to gamma rays than AG1522 cells. For a given dose, GM02052 cells contained a much higher frequency of deletions and misrejoining than AG1522 cells. For both cell types, a good correlation was found between the percentage of aberrant cells and cell survival. The average number of color junctions, which represent the frequency of chromosome misrejoining, was also found to correlate well with survival. However, in a similar surviving population of GM02052 and AG1522 cells, induced by 1 Gy and 6 Gy, respectively, AG1522 cells contained four times more color junctions and half as many deletions as GM02052 cells. These results indicate that both repair deficiency and misrepair may be involved in the hyper-radiosensitivity of AT cells.

  5. Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence in Situ Hybridization by Quantitative Real-Time PCR.

    PubMed

    Koudelakova, Vladimira; Berkovcova, Jitka; Trojanec, Radek; Vrbkova, Jana; Radova, Lenka; Ehrmann, Jiri; Kolar, Zdenek; Melichar, Bohuslav; Hajduch, Marian

    2015-07-01

    Administration of drugs targeting HER2 (official symbol ERBB2) is an important component of therapy for breast cancer patients with HER2 amplification/overexpression as determined by in situ hybridization (ISH) and immunohistochemistry (IHC). In approximately 5% of breast cancers, ISH assays fail. In these cases, HER2 protein expression is evaluated by IHC alone that may yield false negatives/positives for poor-quality samples. Therefore, we developed a method that was based on quantitative real-time PCR applicable for DNA from formalin-fixed, paraffin-embedded tissue samples. Its limit of detection was determined with breast cancer cell lines and validated with 223 breast cancer patient samples. On the basis of comparisons with fluorescent ISH (FISH) and IHC data, the sensitivity of the new method was 94.2% and 95.1%, its specificity was 100% and 99.1%, and overall concordance between results obtained with the quantitative real-time PCR method and FISH/IHC was 97.6% for both methods. The quantitative real-time PCR method was then used to evaluate the HER2 status of 198 of 3696 breast cancer tissues that yielded indeterminate FISH results. The HER2 copy number was successfully determined in 69.2% of these indeterminate samples. Thus, the DNA-based technique appears to be a specific, sensitive method for determining HER2 copy numbers when the FISH assay fails, which may complement IHC tests. PMID:25956448

  6. Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells

    SciTech Connect

    Lemieux, N. ); Malfoy, B. ); Forrest, G.L. )

    1993-01-01

    Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.

  7. Tracking and quantification of nitrifying bacteria in biofilm and mixed liquor of a partial nitrification MBBR pilot plant using fluorescence in situ hybridization.

    PubMed

    Abzazou, Tarik; Araujo, Rosa M; Auset, María; Salvadó, Humbert

    2016-01-15

    A moving bead biofilm reactor (MBBR) pilot plant was implemented as a partial nitrification process for pre-treatment of ammonium-rich liquors (676±195mgL(-1)), and studied for 479days under variations in hydraulic retention time. The main purpose of this work, was the study of dynamics abundance of total bacteria and single-cells nitrifying bacteria belonging to ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in biofilms and mixed liquor of the plant. The microbial monitoring was successfully achieved using fluorescence in situ hybridization combined with flocs disaggregation protocol as a useful microbial monitoring tool. A partial nitrification process with a N-NH4(+) removal rate of about 38.6±14.8% was successfully achieved at 211days after start-up, with a clear dominance of AOB, which accounted for 11.3±17.0% of total bacterial cells compared with only 2.1±4.0% of NOB. The effluent obtained was subsequently supplied to an Anammox reactor for complete ammonium treatment. PMID:26473713

  8. Aneuploidy in 165,330 human sperm; results of two- and three-colour fluorescence in situ hybridization for chromosomes 1, 12, 15, 18, X, and Y

    SciTech Connect

    Spriggs, E.L.; Martin, R.H. |

    1994-09-01

    To understand the mechanisms that affect aneuploidy, fluorescence in situ hybridization (FISH), using chromosome-specific centromeric probes, was employed to screen a large population of human sperm for numerical errors. To determine the true rate of disomy for chromosomes 1, 12, 15, 18, two-color FISH was performed, and for the gonosomes, three-color FISH. The use of multiple, differently-colored probes allows one to distingish a true disomic sperm from a diploid cell. For each centromeric probe, a minimum of 10,000 sperm nuclei for each of five donors was scored, giving a total count of 165,330 sperm nuclei. The incidence of disomic sperm for the sex chromosomes was significantly increased as compared to the frequency for the autosomes ({chi}{sup 2}=232.3, p<0.001), confirming the results observed in studies of sperm karyotypes and spontaneous abortions. The disomy frequencies for autosomes 1, 12, 15, and 18 were found to be uniform. Inter-donor heterogeneity for disomy frequencies was found to exist for the sex chromosomes and for chromosomes 1 and 15, suggesting significant variation among normal men.

  9. Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

    PubMed Central

    Franks, Alison H.; Harmsen, Hermie J. M.; Raangs, Gerwin C.; Jansen, Gijsbert J.; Schut, Frits; Welling, Gjalt W.

    1998-01-01

    Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 1010 cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 × 1010 cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 107 to 7 × 108 per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora. PMID:9726880

  10. Assignment of the human homologue of the mTRiC-P5 gene (TRIC5) to band 1q23 by fluorescence in situ hybridization

    SciTech Connect

    Sevigny, G.; Joly, E.C.; Bibor-Hardy, V.

    1994-08-01

    The TCP1 ring complex (TRiC) is a molecular chaperone involved in actin and tubulin folding. Little is known about the components of this complex. The first component identified was TCP1, a protein coded by a gene in the t-complex locus on mouse chromosome 17. This locus is involved in several embryonic defects, male sterility, and the transmission ratio distortion. In humans, the t-complex genes map to chromosome 6. Other components of TRiC are thought to be TCP1-related proteins. Recently, a mouse cDNA coding for one of these proteins has been cloned and named mTRiC-P5. Here we report the cloning of a partial human cDNA clone, homologous to mTRiC-P5, and its chromosome localization by fluorescence in situ hybridization. The human TRiC-P5 gene (TRIC5) maps to human chromosome 1q23, a region known to be a preferential chromosomal breakpoint involved in leukemia. Therefore, even if TCP1 and TRiC-P5 are related proteins and are found in the same protein complex, they are not coded by syntenic genes in humans. 16 refs., 2 figs.

  11. Immunohistochemistry and Fluorescence In Situ Hybridization Can Inform the Differential Diagnosis of Low-Grade Noninvasive Urothelial Carcinoma with an Inverted Growth Pattern and Inverted Urothelial Papilloma

    PubMed Central

    Lu, Yong-Ming; Zhang, Hui-Zhi; Wang, Tao; Yang, Xiao-Qun; Sun, Meng-Hong; Wang, Chao-Fu

    2015-01-01

    Urothelial carcinoma (UC) comprises a heterogeneous group of epithelial neoplasms with diverse biological behaviors and variable clinical outcomes. Distinguishing UC histological subtypes has become increasingly important because prognoses and therapy can dramatically differ among subtypes. In clinical work, overlapping morphological findings between low-grade noninvasive UC (LGNUC), which exhibits an inverted growth pattern, and inverted urothelial papilloma (IUP) can make subclassification difficult. We propose a combination of immunohistochemistry (IHC) and molecular cytogenetics for subtyping these clinical entities. In our study, tissue microarray immunohistochemical profiles of Ki-67, p53, cytokeratin 20 (CK20) and cyclinD1 were assessed. Molecular genetic alterations such as the gain of chromosomes 3, 7 or 17 or the homozygous loss of 9p21 were also assessed for their usefulness in differentiating these conditions. Based on our analysis, Ki-67 and CK20 may be useful for the differential diagnosis of these two tumor types. Fluorescence in situ hybridization (FISH) can also provide important data in cases in which the malignant nature of an inverted urothelial neoplasm is unclear. LGNUC with an inverted growth pattern that is negative for both Ki-67 and CK20 can be positively detected using FISH. PMID:26208279

  12. Fluorescence in situ hybridization mapping of human chromosome 19: mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting.

    PubMed

    Trask, B; Christensen, M; Fertitta, A; Bergmann, A; Ashworth, L; Branscomb, E; Carrano, A; Van Den Engh, G

    1992-09-01

    Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested). PMID:1330881

  13. Institutional quality assurance for breast cancer HER2 immunohistochemical testing: identification of outlier results and impact of simultaneous fluorescence in situ hybridization cotesting.

    PubMed

    Green, Ian F; Zynger, Debra L

    2015-12-01

    The College of American Pathologists Accreditation Checklist requires comparison of laboratory predictive results with published benchmarks but does not require analysis of individual pathologists. With the availability of targeted human epidermal growth factor receptor 2 (HER2) protein therapy, uniform reporting of HER2 protein status by immunohistochemistry (IHC) is essential. Our aim was to compare HER2 IHC results among pathologists in routine clinical practice within a single institution and assess the impact of simultaneous IHC and fluorescence in situ hybridization (FISH) ordering. We reviewed reports from 928 consecutive breast needle biopsies from 2008 to 2012 at a tertiary academic medical center in which HER2 IHC and HER2 FISH were ordered. There was a significant association between breast pathologist and IHC result (negative, 49.8%-83.2%; positive, 8.7%-14.1%; equivocal, 5.2%-41.5%; P < .0001) but not breast pathologist and FISH result (P = .69). For 1 pathologist, IHC signed out with FISH had an equivocal rate nearly 2-fold lower than IHC results that were reported first (10.5% versus 20.9%) (P = .04). Institutions should be aware that although overall HER2 IHC reporting may be consistent with guidelines, there can be significant variation among practitioners. In addition to aggregate data, we recommend comparing the rates from individual pathologists to standards. Furthermore, routine simultaneous ordering of both IHC and FISH could impact interpretation of test results and may inappropriately encourage less confidence in IHC results among pathologists. PMID:26412217

  14. Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization

    PubMed Central

    Gu, JunGang; Li, LiHua; Zhou, ZhiGang

    2012-01-01

    Background There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (?=?Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination. Methodology/Principal Findings To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4?, 6-diamidino-2-phenylindole (DAPI). The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes. Conclusions/Significance Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future. PMID:23166593

  15. Measuring mRNA copy-number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization (smFISH)

    PubMed Central

    Skinner, Samuel O.; Sepúlveda, Leonardo A.; Xu, Heng; Golding, Ido

    2014-01-01

    We present a method for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently-labeled oligonucleotide probes are hybridized to the target mRNA, so that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The number of target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete “spots” as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy-number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 days, with another 2-3 days typically required for image and data analysis. PMID:23680982

  16. Enhancing the in vitro and in vivo detection of aneuploidy by fluorescence in situ hybridization with the use of bromodeoxyuridine as a proliferation marker.

    PubMed

    Balakrishnan, S; Payawal, J; Schuler, M J; Hasegawa, L; Eastmond, D A

    2002-11-26

    Aneuploidy is associated with spontaneous abortions, birth defects, and many types of human cancers. Currently there are few assays developed for the efficient detection of aneuploidy in vivo. However, with the recent availability of chromosome-specific DNA probes for the rat, fluorescence in situ hybridization (FISH) techniques could be used for the rapid and sensitive detection of aneuploidy in different tissue and cell types. In order to develop a system that can detect alterations in chromosome number in rat cells in vitro, we treated cultured rat lymphocytes with three aneugens-noscapine hydrochloride (0-150 microM) and vincristine and vinblastine sulfate (0-0.06 microM). 5-Bromo-2-deoxyuridine (BrdU; 1 microM) was added to the culture medium to allow proliferating and non-proliferating cells to be distinguished. To test this assay under in vivo conditions, 21-day-old male Sprague-Dawley rats were subcutaneously implanted with osmotic pumps that delivered BrdU (approximately 12 mg/kg per day) continuously. These rats were administered vinblastine sulfate (0, 0.5 and 1mg/kg) by intraperitoneal injection. The rat lymphocytes and hepatocytes incorporating BrdU were detected by immuno-fluorescent labeling, and FISH with a rat chromosome 4 probe was performed on the labeled and unlabeled cells. Highly significant increases in hyperdiploidy were seen in the replicating rat lymphocytes treated with noscapine, vincristine or vinblastine in vitro and in the rat hepatocytes treated with vinblastine in vivo. In contrast, no significant increase in hyperdiploidy was observed in the non-replicating cells. These results demonstrate that this BrdU-enhanced FISH assay with chromosome-specific rat probes can be used to efficiently detect numerical chromosomal aberrations in vitro and in vivo in slowly or moderately replicating rat tissues. The combination of BrdU-labeling and FISH allows the scoring of hyperdiploidy to be focused on the actively replicating cells, thereby increasing the sensitivity of the FISH technique. PMID:12438006

  17. Concurrent use of flow cytometry and fluorescence in-situ hybridization techniques for detecting faulty meiosis in a human sperm sample.

    PubMed

    Weissenberg, R; Aviram, A; Golan, R; Lewin, L M; Levron, J; Madgar, I; Dor, J; Barkai, G; Goldman, B

    1998-01-01

    Routine semen analysis in an infertile patient revealed severe teratospermia associated with malformation of head and tail in 100% of the sperm cells. Flow cytometry and fluorescence in-situ hybridization (FISH) were shown to supplement routine semen analysis by providing information on the sperm chromatin. Using flow cytometry, propidium iodide-stained spermatozoa from the same sperm sample were compared with a normal reference pool, and with human lymphocytes. The results point to a population of diploid sperm cells rather than to mature haploid spermatozoa. Numerical chromosomal abnormalities of the spermatozoa were subsequently evaluated using FISH. A total of 1000 sperm cells were scored for X and Y chromosomes, and an additional 1128 sperm cells for chromosome 18. Aneuploidy of chromosomes X and Y was revealed in 96.9% of the cells and of chromosome 18 in 90.3% of the cells. Non-disjunction of chromosome X and Y in meiosis I and II occurred in 54.8 and 2.7% of the sperm cells respectively. Non-disjunction in both meiosis I and II occurred in 39.4% of the sperm cells. A normal haploid pattern for chromosomes X and Y was observed in only 3.1%, and for chromosome 18 in 9.7%, of the cells. Using three colour FISH for the sex chromosomes and for chromosome 18, diploidy was demonstrated in 19.4% of 500 sperm cells and aneuploidy in virtually all sperm cells (99.2%). The use of flow cytometry and FISH in cases where genetic and developmental chromatin abnormalities are suspected is a valuable adjunct to other available techniques, and can guide the clinicians to decide which samples are unsuitable for intracytoplasmic injection. PMID:9510012

  18. Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing.

    PubMed

    Walther, Charles; Hofvander, Jakob; Nilsson, Jenny; Magnusson, Linda; Domanski, Henryk A; Gisselsson, David; Tayebwa, Johnbosco; Doyle, Leona A; Fletcher, Christopher D M; Mertens, Fredrik

    2015-09-01

    Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNA-Seq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues. PMID:26121314

  19. Definition of a fluorescence in-situ hybridization score identifies high- and low-level FGFR1 amplification types in squamous cell lung cancer.

    PubMed

    Schildhaus, Hans-Ulrich; Heukamp, Lukas C; Merkelbach-Bruse, Sabine; Riesner, Katharina; Schmitz, Katja; Binot, Elke; Paggen, Ellen; Albus, Kerstin; Schulte, Wolfgang; Ko, Yon-Dschun; Schlesinger, Andreas; Ansén, Sascha; Engel-Riedel, Walburga; Brockmann, Michael; Serke, Monika; Gerigk, Ulrich; Huss, Sebastian; Göke, Friederike; Perner, Sven; Hekmat, Khosro; Frank, Konrad F; Reiser, Marcel; Schnell, Roland; Bos, Marc; Mattonet, Christian; Sos, Martin; Stoelben, Erich; Wolf, Jürgen; Zander, Thomas; Buettner, Reinhard

    2012-11-01

    We recently reported fibroblast growth factor receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ?2.0, or average number of FGFR1 signals per tumor cell nucleus ?6, or the percentage of tumor cells containing ?15 FGFR1 signals or large clusters ?10%) was detected at a frequency of 16% and low-level amplification (as defined by ?5 FGFR1 signals in ?50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials. PMID:22684217

  20. Nucleic acid in-situ hybridization detection of infectious agents

    NASA Astrophysics Data System (ADS)

    Thompson, Curtis T.

    2000-04-01

    Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

  1. Gene amplification of ESR1 in breast cancers--fact or fiction? A fluorescence in situ hybridization and multiplex ligation-dependent probe amplification study.

    PubMed

    Ooi, Akishi; Inokuchi, Masafumi; Harada, Shinichi; Inazawa, Johji; Tajiri, Ryousuke; Kitamura, Seiko Sawada-; Ikeda, Hiroko; Kawashima, Hiroko; Dobashi, Yoh

    2012-05-01

    Oestrogen receptor-alpha (ER?), encoded by the ESR1 gene located on 6q25, is a nuclear transcription factor. Since it was reported in 2007 that more than 20% of breast cancers show ESR1 gene amplification, there has been considerable controversy about its frequency and clinical significance. We set out to assess the frequency and levels of ESR1 amplification in breast cancers. In a total of 106 breast needle biopsy specimens examined by immunohistochemistry, 78 tumours contained more than 10% ER?-positive cancer cells. In fluorescence in situ hybridization (FISH) analysis with an ESR1-specific probe, variously extended ESR1 signals were found in ER?-expressing cells. Some of these were indistinguishable from large clustered signals generally accepted to mean high-level gene amplification in homogeneously staining regions (HSRs), and could be considered to represent gene amplification. However, with RNase treatment, the 'HSR-like' signals changed to small compact signals, and are thus thought to represent concentrated RNA. FISH using two differently labelled probes corresponding to the non-overlapping 5'- and 3'-end portions of the ESR1 gene on touch smears showed a preserved spatial relationship of the 3' to 5' sequence of ESR1, therefore strongly suggesting that the RNA consisted of primary transcripts. Using touch smears obtained from 51 fresh tumours, precise enumeration of ESR1 signals with a correction by the number of centromere 6 on FISH after RNase A treatment revealed that three tumours (5.9%) had tumour cells with one to three additional copies of ESR1 as predominant subpopulations. This infrequent and low level of gene amplification of ESR1 was also detected as a 'gain' of the gene by analysis with multiplex ligation-dependent probe amplification (MLPA). The consistent results from immunohistochemistry, FISH, and MLPA in the present study settle the long-standing debate concerning gene amplification of ESR1 in breast carcinoma. PMID:22170254

  2. Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

    PubMed Central

    Cerqueira, Laura; Fernandes, Ricardo M.; Ferreira, Rui M.; Oleastro, Mónica; Carneiro, Fátima; Brandão, Catarina; Pimentel-Nunes, Pedro; Dinis-Ribeiro, Mário; Figueiredo, Céu; Keevil, Charles W.; Vieira, Maria J.

    2013-01-01

    Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach. PMID:23596234

  3. Evidence of Multiple Treponema Phylotypes Involved in Bovine Digital Dermatitis as Shown by 16S rRNA Gene Analysis and Fluorescence In Situ Hybridization?

    PubMed Central

    Klitgaard, Kirstine; Boye, Mette; Capion, Nynne; Jensen, Tim K.

    2008-01-01

    The etiopathogenesis of the skin disease digital dermatitis (DD), an important cause of lameness in cattle, remains uncertain. Microscopically, the disease appears to be polymicrobial, with spirochetes as the predominant bacteria. The objective of this study was to identify the main part of the bacteria involved in DD lesions of cattle by using culture-independent molecular methods. Ten different phylotypes of Treponema were identified either by 16S rRNA gene sequencing of bacteria from DD lesions or by fluorescence in situ hybridization (FISH) analysis using phylotype-specific 16S rRNA-directed oligonucleotide probes. Two phylotypes, phylotype 1 (PT1) and PT2, were not closely related to any characterized treponemal species. PT7 was 99.3% identical to Treponema denticola, while PT9 resembled T. vincentii by 96%. The remaining phylotypes, PT3, PT4, PT5, PT6, and PT8, and Treponema brennaborense had previously been isolated from DD lesions. Forty DD biopsy specimens were examined for Treponema by FISH. With one exception, all of the biopsy specimens revealed epidermotropic, intermingled infection with three or more different phylotypes (mean, 4.7). The most prevalent species were PT1 (95%), PT6 (93%), and PT3 (85%). While colonization by PT3 was confined to the surface of the epidermis, both PT1 and PT6 invaded deep into the stratum spinosum and were seen in ulcerated dermal papillae. In two cases, all 10 phylotypes were demonstrated. Furthermore, FISH with a Treponema group-specific probe showed that Treponema accounted for more than 90% of the total bacterial population in the biopsy specimens. These data strongly suggest that a group of apparently symbiotic Treponema species are involved as primary bacterial pathogens in DD. PMID:18562583

  4. Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples.

    PubMed

    Almeida, C; Azevedo, N F; Fernandes, R M; Keevil, C W; Vieira, M J

    2010-07-01

    A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 x 10(9) +/- 5 x 10(8) CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 x 10(7) +/- 5 x 10(6) CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity. PMID:20453122

  5. Reliability Evaluation of Fluorescence In Situ Hybridization (FISH) and G-Banding on Bone Marrow and Peripheral Blood Cells in Chronic Myelogenous Leukemia Patients.

    PubMed

    Manaflouyan Khajehmarjany, Soheila; Rahmani, Seyed Ali; Chavoshi, Seyed Hadi; Esfahani, Ali; Movassaghpour Akbari, Ali Akbar

    2015-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disease. The cytogenetic hallmark of CML is Philadelphia (Ph) chromosome. This study aimed to diagnose suspected CML patients, to monitor CML patients under therapy using cytogenetic and fluorescence in situ hybridization (FISH) techniques to analyze their bone marrow (BM) and peripheral blood (PB) samples, and finally to compare their obtained results for both specimens. This study was conducted during one-year period (2012-2013). The participants were recruited from the Hematology and Oncology Clinic of Shahid Gazi (Emam Reza) Hospital of Tabriz University of Medical Sciences, Tabriz, East Azerbaijan Province, Iran. We analyzed 90 samples from 60 suspected CML patients (30 BM and 60 PB samples). All samples were analyzed using G-banding, 5 samples using dual fusion FISH (DF-FISH) probes, as well as 30 samples using both FISH and G-banding. Among the 90 analyzed samples of 60 patients, 25 (41.66%) were Ph+ using karyotyping, whereas five cases were not analyzable, so FISH was applied and the results confirmed that only two individuals were BCR-ABL+. In the comparison between 25 BM and 25 PB samples using karyotyping, 15 (60%) and 10 (40%) were ph+, respectively. The comparison of FISH and karyotyping on 30 samples showed that 9 (30%) and 8 (26.66%) were Ph+, respectively, and only 18.18% of Ph+ patients showed atypical patterns. In the comparison between BM-cytogenetic and PB- interphase-FISH (I-FISH), BM-cytogenetic was more reliable than PB-I-FISH in detecting Ph. Our data demonstrate that FISH analysis is a rapid, reliable and sensitive technique. The comparison between BM and PB showed that PB can not be replaced by BM, even in detecting by FISH. PMID:25870848

  6. Next-Generation Sequencing and Fluorescence in Situ Hybridization Have Comparable Performance Characteristics in the Analysis of Pancreaticobiliary Brushings for Malignancy.

    PubMed

    Dudley, Jonathan C; Zheng, Zongli; McDonald, Thomas; Le, Long P; Dias-Santagata, Dora; Borger, Darrell; Batten, Julie; Vernovsky, Kathy; Sweeney, Brenda; Arpin, Ronald N; Brugge, William R; Forcione, David G; Pitman, Martha B; Iafrate, A John

    2016-01-01

    Cytological evaluation of pancreatic or biliary duct brushings is a specific, but insensitive, test for malignancy. We compared adjunctive molecular testing with next-generation sequencing (NGS) relative to fluorescence in situ hybridization (FISH) for detection of high-risk neoplasia or malignancy. Bile duct brushings from 81 specimens were subjected to cytological analysis, FISH using the UroVysion probe set, and targeted NGS. Specimens were placed into negative/atypical (negative) or suspicious/positive (positive) categories depending on cytology and negative or positive categories on the basis of FISH and NGS results. Performance characteristics for each diagnostic modality were calculated on the basis of clinicopathologic follow-up and compared in a receiver operating characteristic analysis. There were 33 high-risk neoplasia/malignant strictures (41%) and 48 benign (59%). NGS revealed driver mutations in 24 cases (30%), including KRAS (21 of 24 cases), TP53 (14 of 24 cases), SMAD4 (6 of 24 cases), and CDKN2A (4 of 24 cases). Cytology had a sensitivity of 67% (95% CI, 48%-82%) and a specificity of 98% (95% CI, 89%-100%). When added to cytology, NGS increased the sensitivity to 85% (95% CI, 68%-95%), leading to a significant increase in the area under the curve in a receiver operating characteristic analysis (P = 0.03). FISH increased the sensitivity to 76% (95% CI, 58%-89%), without significantly increasing the area under the curve. These results suggest that ancillary NGS testing offers advantages over FISH, although studies with larger cohorts are needed to verify these findings. PMID:26596524

  7. Evidence for Cytogenetic and Fluorescence In Situ Hybridization Risk Stratification of Newly Diagnosed Multiple Myeloma in the Era of Novel Therapies

    PubMed Central

    Kapoor, Prashant; Fonseca, Rafael; Rajkumar, S. Vincent; Sinha, Shirshendu; Gertz, Morie A.; Stewart, A. Keith; Bergsagel, P. Leif; Lacy, Martha Q.; Dingli, David D.; Ketterling, Rhett P.; Buadi, Francis; Kyle, Robert A.; Witzig, Thomas E.; Greipp, Philip R.; Dispenzieri, Angela; Kumar, Shaji

    2010-01-01

    Overall survival (OS) has improved with increasing use of novel agents in multiple myeloma (MM). However, the disease course remains highly variable, and the heterogeneity largely reflects different genetic abnormalities. We studied the impact of the Mayo risk-stratification model of MM on patient outcome in the era of novel therapies, evaluating each individual component of the model—fluorescence in situ hybridization (FISH), conventional cytogenetics (CG), and the plasma cell labeling index—that segregates patients into high- and standard-risk categories. This report consists of 290 patients with newly diagnosed MM, predominantly treated with novel agents, who were risk-stratified at diagnosis and were followed up for OS. Of these patients, 81% had received primarily thalidomide (n=50), lenalidomide (n=199), or bortezomib (n=79) as frontline or salvage therapies. Our retrospective analysis validates the currently proposed Mayo risk-stratification model (median OS, 37 months vs not reached for high- and standard-risk patients, respectively; P=.003). Although the FISH or CG test identifies a high-risk cohort with hazard ratios of 2.1 (P=.006) and 2.5 (P=.006), respectively, the plasma cell labeling index cutoff of 3% fails to independently prognosticate patient risk (hazard ratio, 1.4; P=.41). In those stratified as standard-risk by one of the 2 tests (FISH or CG), the other test appears to be of additional prognostic significance. This study validates the high-risk features defined by FISH and CG in the Mayo risk-stratification model for patients with MM predominantly treated with novel therapies based on immunomodulatory agents. PMID:20511484

  8. Identification of a microdeletion at 7q21.3 with fluorescence in situ hybridization in a patient with split hand/split foot (ectrodactyly)

    SciTech Connect

    Hudgins, L.; Massa, H.; Disteche, C.

    1994-09-01

    Split hand/split foot (SHSF), often referred to as ectrodactyly or lobster claw deformity, is a human developmental disorder characterized by a deep median cleft of the hands and feet, missing digits, and fusion of remaining digits. This anomaly can be seen alone, frequently autosomal dominant, or in association with other abnormalities. One locus for this defect has been localized to chromosome 7q21.3-q22.1. We report a patient with SHSF plus mental retardation, short stature and dysmorphic features who was found to have a microdeletion at this locus detected only with the aid of fluorescence in situ hybridization (FISH). T.H. is a 7 y.o. male who was referred for evaluation of foot anomalies and mild mental retardation. History was remarkable for growth retardation of postnatal onset and hypotonia. Renal ultrasound and audiology evaluation were normal. Physical exam revealed dysplastic ears, micrognathia, long philtrum, high narrow palate, and malformations of the feet consistent with SHSF. Family history was negative for limb abnormalities and mental retardation. A number of patients with SHSF and other anomalies have been found to have deletions involving chromosome 7q21-q22; therefore, high resolution chromosome analysis was performed in T.H. but was inconclusive. Cosmids and yeast artificial chromosomes which we had previously mapped to the SHSF critical region were used as FISH probes and a microdeletion was detected. We were thus able to determine the etiology of this child`s abnormalities and provide accurate genetic counseling, which would not have been possible with standard cytogenetic techniques. This technique also allowed us to further refine the SHSF critical region. This case illustrates the utility of FISH for the rapid identification of suspect microdeletions in SHSF. This approach should also be useful as an expeditious way of defining the critical regions for the location of genes which give rise to other developmental malformations.

  9. The human gene for xeroderma pigmentosum complementation group G (XPG) maps to 13q33 by fluorescence in situ hybridization

    SciTech Connect

    Samec, S.; Corlet, J.; Scherly, D.; Clarkson, S.G. ); Jones, T.A.; Sheer, D. ); Wood, R.D. )

    1994-05-01

    Recently, a human cDNA was isolated that restores normal levels of UV resistance and DNA repair synthesis when expressed in vivo in a lymphoblastoid cell line representing XP group G. The XP-G complementing gene (XPG) generates an mRNA of [approximately]4 kb and encodes a protein (XPGC) with homology to the RAD2 DNA repair protein of Saccharomyces cerevisiae. One hundred twenty nanograms of labeled probe was mixed with 2 [mu]g of human C[sub 0]t-1 DNA as a competitor for repetitive elements. Denaturation, dehydration, hybridization, and washing were performed as described. Probe detection was achieved by incubating metaphase spreads sequentially with 2 [mu]g/ml avidin-Texas Red, 5 [mu]g/ml biotinylated goat anti-avidin antibody, and 2 [mu]g/ml avidin-Texas Red. R-banding was revealed after incubation with fluorescein-labeled anti-BrdU mouse monoclonal antibody. Chromosomes were counter-stained with 0.06 [mu]g/ml DAPI in Citifluor. Analysis of 40 metaphase spreads showed paired signals on both copies of chromosome 13 at band 13q33. No other paired signals were seen consistently on any other chromosome. 9 refs., 1 fig.

  10. Large-scale polymorphism near the ends of several human chromosomes analyzed by using fluorescence in situ hybridization (FISH)

    SciTech Connect

    Trask, B.J.; Friedman, C.; Giorgi, D.

    1994-09-01

    We have discovered a large DNA segment that is polymorphically present at the ends of several human chromosomes. The segment, f7501, was originally derived form a human chromosome 19-specific cosmid library. FISH was used to determine the cosmid`s chromosomal distribution on 44 unrelated humans and several closely related primates. The human subjects represent a diversity of reproductively isolated ethnic populations. FISH analysis revealed that sequences highly homologous to the cosmid`s insert are present on both homologs at 3q, 15q,. and 19p in almost all individuals (88, 85, and 87 of 88 homologs, respectively). Other chromosomes sites were labeled much more rarely in the sampled individuals. For example, 56 of the 88 analyzed chromosomes 11 were labeled (18+/+, 6-/-, and 20+/- individuals). In contrast, 2q was labeled on only 1/88 sampled chromosomes. The termini of 2q, 5q, 6p, 6q, 7p, 8p, 9p, 9q, 11p, 12q, 16p, 19q, and 20q and an interstitial site at 2q13-14 were labeled in at least one individual of the set. EcoR1-fragments derived from the cosmid showed the same hybridization pattern as the entire cosmid, indicating that at least 40 kbp is shared by these chromosome ends. Ethnic differences in the allele frequency of these polymorphic variants was observed. For example, signals were observed on 8/10 and 7/10 of the chromosomes 7p and 16q, respectively, derived form Biakan Pygmies, but these sites were infrequently labeled in non-Pygmy human populations (2/68, respectively). This region has undergone significant changes in chromosome location during human evolution. Strong signal was seen on chimpanzee and gorilla chromosome 3, which is homologous to human chromosome 4, a chromosome unlabeled in any of the humans we have analyzed.

  11. Targeting the Treponemal Microbiome of Digital Dermatitis Infections by High-Resolution Phylogenetic Analyses and Comparison with Fluorescent In Situ Hybridization

    PubMed Central

    Foix Bretó, Antoni; Boye, Mette; Jensen, Tim K.

    2013-01-01

    Modern pyrosequencing technology allows for a more comprehensive approach than traditional Sanger sequencing for elucidating the etiology of bovine digital dermatitis. We sought to describe the composition and diversity of treponemes in digital dermatitis lesions by using deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with species-level taxonomic identification. Treponema-specific 16S rRNA gene PCRs and pyrosequencing were performed on biopsy specimens originating from 10 different Catalan dairy herds (n = 36) with digital dermatitis, and this analysis yielded 75,297 sequences. We identified 20 different taxa, including a potentially novel phylotype that displayed 95% sequence identity to members of the Treponema denticola/Treponema pedis-like cluster. Species frequencies and abundances that were determined by pyrosequencing analysis were highly correlated with the results of fluorescent in situ hybridization using phylotype-specific oligonucleotide probes. In a limited number of animals from a single geographic region, we detected most of the Treponema phylotypes that were described in previous investigations of digital dermatitis. Additionally, we identified a number of phylotypes that mapped to oral treponemes of humans and dogs that had not been reported for digital dermatitis lesions. The results presented here support previous observations of a polytreponemal etiology of infections, with Treponema phagedenis-like, Treponema medium/Treponema vincentii-like, and T. denticola/T. pedis-like phylotypes being highly associated with disease. Using this new approach, it has become feasible to study large herds and their surrounding environments, which might provide a basis for a better understanding of the pathogenesis of this disease. PMID:23658264

  12. Genomic In Situ Hybridization (GISH) as a Tool to Identify Chromosomes of Parental Species in Sunflower Interspecific Hybrids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interspecific hybridization has been widely used to transfer genes from wild species into cultivated sunflower. Fluorescent genomic in situ hybridization (GISH) has been used to identify alien chromosomes or segments in other crops, but an equivalent technique for sunflower is lacking. The objective...

  13. Karyotype analysis and visualization of 45S rRNA genes using fluorescence in situ hybridization in aroids (Araceae)

    PubMed Central

    Lakshmanan, Prabhu Shankar; Van Laere, Katrijn; Eeckhaut, Tom; Van Huylenbroeck, Johan; Van Bockstaele, Erik; Khrustaleva, Ludmila

    2015-01-01

    Abstract Karyotype analysis and FISH mapping using 45S rDNA sequences on 6 economically important plant species Anthurium andraeanum Linden ex André, 1877, Monstera deliciosa Liebmann, 1849, Philodendron scandens Koch & Sello, 1853, Spathiphyllum wallisii Regel, 1877, Syngonium auritum (Linnaeus, 1759) Schott, 1829 and Zantedeschia elliottiana (Knight, 1890) Engler, 1915 within the monocotyledonous family Araceae (aroids) were performed. Chromosome numbers varied between 2n=2x=24 and 2n=2x=60 and the chromosome length varied between 15.77 µm and 1.87 µm. No correlation between chromosome numbers and genome sizes was observed for the studied genera. The chromosome formulas contained only metacentric and submetacentric chromosomes, except for Philodendron scandens in which also telocentric and subtelocentric chromosomes were observed. The highest degree of compaction was calculated for Spathiphyllum wallisii (66.49Mbp/µm). B-chromosome-like structures were observed in Anthurium andraeanum. Their measured size was 1.87 times smaller than the length of the shortest chromosome. After FISH experiments, two 45S rDNA sites were observed in 5 genera. Only in Zantedeschia elliottiana, 4 sites were seen. Our results showed clear cytogenetic differences among genera within Araceae, and are the first molecular cytogenetics report for these genera. These chromosome data and molecular cytogenetic information are useful in aroid breeding programmes, systematics and evolutionary studies. PMID:26140158

  14. The comparative utility of fluorescence in situ hybridization and reverse transcription-polymerase chain reaction in the diagnosis of alveolar rhabdomyosarcoma.

    PubMed

    Thway, Khin; Wang, Jayson; Wren, Dorte; Dainton, Melissa; Gonzalez, David; Swansbury, John; Fisher, Cyril

    2015-08-01

    Fluorescence in situ hybridization (FISH) for FOXO1 gene rearrangement and reverse transcription-polymerase chain reaction (PCR) for PAX3/7-FOXO1 fusion transcripts have become routine ancillary tools for the diagnosis of alveolar rhabdomyosarcomas (ARMS). Here we summarize our experience of these adjunct diagnostic modalities at a tertiary center, presenting the largest comparative series of FISH and PCR for suspected or possible ARMS to date. All suspected or possible ARMS tested by FISH or PCR for FOXO1 rearrangement or PAX3/7-FOXO1 fusion transcripts over a 7-year period were included. FISH and PCR results were correlated with clinical and histologic findings. One hundred samples from 95 patients had FISH and/or PCR performed. FISH had higher rates of technical success (96.8 %) compared with PCR (88 %). Where both tests were utilized successfully, there was high concordance rate between them (94.9 %). In 24 histologic ARMS tested for FISH or PCR, 83.3 % were translocation-positive (all for PAX3-FOXO1 by PCR) and included 3 histologic solid variants. In 76 cases where ARMS was excluded, there were 3 potential false-positive cases with FISH but none with PCR. PCR had similar sensitivity (85.7 %) and better specificity (100 %) in aiding the diagnosis of ARMS, compared with FISH (85 and 95.8 %, respectively). All solid variant ARMS harbored FOXO1 gene rearrangements and PAX3-FOXO1 ARMS were detected to the exclusion of PAX7-FOXO1. In comparative analysis, both FISH and PCR are useful in aiding the diagnosis of ARMS and excluding its sarcomatous mimics. FISH is more reliable technically but has less specificity than PCR. In cases where ARMS is in the differential diagnosis, it is optimal to perform both PCR and FISH: both have similar sensitivities for detecting ARMS, but FISH may confirm or exclude cases that are technically unsuccessful with PCR, while PCR can detect specific fusion transcripts that may be useful prognostically. PMID:25912319

  15. Epidermal growth factor receptor and AKT1 gene copy numbers by multi-gene fluorescence in situ hybridization impact on prognosis in breast cancer

    PubMed Central

    Li, Jiao; Su, Wei; Zhang, Sheng; Hu, Yunhui; Liu, Jingjing; Zhang, Xiaobei; Bai, Jingchao; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lei, Zhenmin; Zhang, Jin

    2015-01-01

    The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes. PMID:25702787

  16. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE

    EPA Science Inventory

    A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligo...

  17. &p.1:Abstract Interphase fluorescence in situ hybridization (FISH) was performed on 15-m-thick paraffin sections

    E-print Network

    Rodenacker, Karsten

    spots) in 18/19 cases, and mono- somy 7 (-7) in 13/19 cases. In 5-µm sections, however, trisomy 7 aberration type. Trisomy 7 (+7) became apparent in 15-µm-thick sections in all 19 tu- mors, polysomy 7 (>3

  18. Cytomolecular characterization of rRNA gene sequences among Citrullus species and subspecies using fluorescent in situ hybridization (FISH) technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In previous studies, many DNA markers showed strong preferential (non-Mendelian) segregation in F2 and BC1 genetic populations derived from crosses between wild type watermelon [C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.] (CLC) and watermelon cultivar...

  19. Novel, rapid DNA-based on-chip bacterial identification system combining dielectrophoresis and amplification-free fluorescent resonance energy transfer assisted in-situ hybridization (FRET-ISH)

    NASA Astrophysics Data System (ADS)

    Packard, Michelle M.; Shusteff, Maxim; Alocilja, Evangelyn

    2011-10-01

    Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in-situ hybridization (FRET-ISH) species identification. Identification is achieved completely on chip in less than thirty minutes from receipt of sample compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

  20. Human cDNA mapping using fluorescence in situ hybridization. Final progress report, April 1, 1994--July 31, 1997

    SciTech Connect

    Korenberg, J.R.

    1997-12-31

    The ultimate goal of this research is to generate and apply novel technologies to speed completion and integration of the human genome map and sequence with biomedical problems. To do this, techniques were developed and genome-wide resources generated. This includes a genome-wide Mapped and Integrated BAC/PAC Resource that has been used for gene finding, map completion and anchoring, breakpoint definition and sequencing. In the last period of the grant, the Human Mapped BAC/PAC Resource was also applied to determine regions of human variation and to develop a novel paradigm of primate evolution through to humans. Further, in order to more rapidly evaluate animal models of human disease, a BAC Map of the mouse was generated in collaboration with the MTI Genome Center, Dr. Bruce Birren.

  1. Phenotypic consequences of a mosaic marker chromosome identified by fluorescence in situ hybridization (FISH) as being derived from chromosome 16

    SciTech Connect

    Ray, J.H.; Zhou, X.; Pletcher, B.A.

    1994-09-01

    De novo marker chromosomes are detected in 1 in 2500 amniotic fluid samples and are associated with a 10-15% risk for phenotypic abnormality. FISH can be utilized as a research tool to identify the origins of marker chromosomes. The phenotypic consequences of a marker chromosome derived from the short arm of chromosome 16 are described. A 26-year-old woman underwent amniocentesis at 28 weeks gestation because of a prenatally diagnosed tetralogy of Fallot. Follow-up ultrasounds also showed ventriculomegaly and cleft lip and palate. 32 of 45 cells had the karyotype 47,XY,+mar; the remaining cells were 46,XY. The de novo marker chromosome was C-band positive and non-satellited and failed to stain with distamycin A/DAPI. At birth the ultrasound findings were confirmed and dysmorphic features and cryptorchidism were noted. Although a newborn blood sample contained only normal cells, mosaicism was confirmed in 2 skin biopsies. FISH using whole-chromosome painting and alpha-satellite DNA probes showed that the marker chromosome had originated from chromosome 16. As proximal 16q is distamycin A/DAPI positive, the marker is apparently derived from proximal 16p. At 15 months of age, this child is hypotonic, globally delayed and is gavage-fed. His physical examination is significant for microbrachycephaly, a round face, sparse scalp hair, ocular hypertelorism, exotropia, a flat, wide nasal bridge and tip, mild micrognathia, and tapered fingers with lymphedema of hands and feet. Inguinal hernias have been repaired. His features are consistent with those described for patients trisomic for most or all of the short arm of chromosome 16. Marker chromosomes derived from the short arm of chromosome 16 appear to have phenotypic consequences. As the origin of more marker chromosomes are identified using FISH, their karyotype/phenotype correlations will become more apparent, which will permit more accurate genetic counseling.

  2. Localization of the human cytoplasmic dynein heavy chain (DNECL) to 14qter by fluorescence in situ hybridization

    SciTech Connect

    Narayan, D.; Ravikumar, T.S.; Desai, T.

    1994-08-01

    Dyneins are a group of microtubule-activated ATPases that serve to convert chemical energy into mechanical energy. They have been divided into two large subgroups, namely the axonemal and cytoplasmic dyneins. Cytoplasmic dynein has been implicated in a variety of other forms of intracellular motility, including retrograde axonal transport, protein sorting between apical and basolateral surfaces, and redistribution of organelles like endosomes and lysosomes. Our report is the first chromosomal localization of the human ctyoplasmic dynein heavy chain (DNECL). 7 refs., 1 fig.

  3. Assignment of the tyrosinase-related protein-2 gene (TYRP2) to human chromosome 13q31-q32 by fluorescence in situ hybridization: Extended synteny with mouse chromosome 14

    SciTech Connect

    Sturm, R.A. ); Baker, E.; Sutherland, G.R. )

    1994-05-01

    A recombinant human genomic liver DNA [lambda]-phage library was screened with the insert of the pHuTRP-2 cDNA clone to isolate a series of bacteriophage with inserts spanning the human TYRP2 gene. One of the [lambda]-phage clones ([lambda]HuT-YRP2-7) containing a 2-kb HindIII fragment with the 5[prime] exon sequence of the cDNA as determined by sequence analysis was used for the gene localization study. DNA prepared from the phage by Qiagen chromatography was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 5 ng/[mu]l to metaphases from two normal males. The fluorescence in situ hybridization method was modified from that previously described in that chromosomes were stained before analysis with both propidium iodide as counterstain and DAPI for chromosome identification. Twenty metaphases from the first normal male were examined for fluorescent signal. All of these metaphases showed signal on one or both chromatids of chromosome 13 in the region 13q31-q33; 88% of this signal was at the interface of bands 13q31-q32. There was a total of four nonspecific background dots observed in these 20 metaphases. A similar result was obtained from hybridization of the probe to 20 metaphases from the second normal male (data not shown). This region has also been shown to contain the propionyl coenzyme A carboxylase [alpha]-chain gene by in situ hybridization. The localization of the TYRP2 locus to human chromosome 13q31-q32 extends the syntenic region of chromosome 13 with mouse chromosome 14. 15 refs., 1 fig.

  4. Le diagnostic anténatal de la trisomie 21 par l'hybridation in situ en fluorescence (FISH): à propos des premiers tests réalisés au Maroc

    PubMed Central

    Lamzouri, Afaf; Natiq, Abdelhafid; Tajir, Mariam; Sendid, Mohamed; Sefiani, Abdelaziz

    2012-01-01

    Introduction Le but de cette étude était de présenter les premiers résultats de diagnostic anténatal de la trisomie 21 par la technique d'hybridation in situ en fluorescence (FISH) au Maroc et discuter son intérêt dans le diagnostic rapide de cette aneuploïdie. Méthodes Ce travail a été réalisé chez 23 femmes avec des grossesses à haut risque de trisomie 21. La moyenne d’âge des gestantes étaient de 37,43 ans avec des extrêmes de 21 et 43 ans. Toutes étaient musulmanes mariées, mariage légitimé par la Charia, dont trois mariages consanguins, sauf une originaire de la République Démocratique du Congo qui était chrétienne et concubine. La majorité des femmes étaient fonctionnaires et avaient un niveau de scolarisation moyen à élevé. Toutes les patientes ont bénéficié d'une consultation de génétique médicale au cours de laquelle il leur a été donné des informations sur la technique, son intérêt et ses limites. Il s'agit de femmes enceintes qui avaient soit un âge maternel élevé ou des signes d'appel échographiques et/ ou biochimiques. Une des patientes était porteuse d'une translocation robertsonienne t(14;21) équilibrée. Une amniocentèse a été réalisée chez toutes les gestantes et aucun avortement n'a était induit par ce geste invasif. L’âge gestationnel moyen à la première consultation était de 14 semaines d'aménorrhée (SA) et à l'amniocentèse était de 16 SA et 5 jours. L'analyse FISH a été réalisée, après consentement des couples, sur des cellules non cultivées à partir des échantillons de liquides amniotiques, en utilisant des sondes spécifiques du chromosome 21. Résultats Parmi les 23 patientes qui ont bénéficiées d'un diagnostic anténatal de la trisomie 21 par la technique FISH, nous avons pu rassurer 21 d'entre elles, et nous avons détecté deux cas de trisomie 21 fœtal. Conclusion La technique FISH permet un diagnostic anténatal rapide, en moins de 48h, de la trisomie 21 sur une faible quantité de liquide amniotique. Elle offre aux couples l'avantage de prendre, dans des délais raisonnables, la décision qui leur convient concernant la poursuite ou non de la grossesse. Elle permet souvent, avec un résultat normal, de rassurer rapidement les femmes enceintes trop angoissées. PMID:23330029

  5. Zebrafish Whole-Mount In Situ Hybridization Followed by Sectioning.

    PubMed

    Doganli, Canan; Nyengaard, Jens Randel; Lykke-Hartmann, Karin

    2016-01-01

    In situ hybridization is a powerful technique used for locating specific nucleic acid targets within morphologically preserved tissues and cell preparations. A labeled RNA or DNA probe hybridizes to its complementary mRNA or DNA sequence within a sample. Here, we describe RNA in situ hybridization protocol for whole-mount zebrafish embryos. PMID:26695046

  6. Detection of {open_quotes}cryptic{close_quotes}karyotypic rearrangements in closely related primate species by fluorescence in situ hybridization (FISH) using human subtelomeric DNA probes

    SciTech Connect

    Youngblom, J.J.; Trask, B.J.; Friedman, C.

    1994-09-01

    Specific human subtelomeric DNA probes were used to reveal cryptic chromosomal rearrangements that cannot be detected by conventional high resolution cytogenetic techniques, or by chromosomal in situ suppression hybridization using whole chromosome paint analysis. Two cosmids containing different subtelomeric DNA sequences were derived from human chromosome 19 and designated as 7501 and 16432. Cosmid 7501 was hybridized to chromosomes from humans, chimpanzee, gorilla and orangutan. In humans, 7501 consistently labeled chromosomes 3q, 15q, and 19p. Additional chromosomes were labeled in different individuals, indicating a polymorphic distribution of this sequence in the human genome. In contrast, 7501 consistently and strongly labeled only the q arm terminus of chromosome 3 in both chimp and gorilla. The identification of the chromosome was made by two-color FISH analysis using human chromosome 4-specific paint and homologous to human chromosome 4. None of the human subjects showed labeling of chromosome 4 with 7501. This finding suggests that in the course of human evolution, subsequent to the divergence of humans and African apes, a cryptic translocation occurred between the ancestral human chromosome 4 and one or more of the other human chromosomes that now contain this DNA segment. In orangutan, 7501 labeled a single acrocentric chromosome pair, a distinctly different chromosome than that labeled in chimp and gorilla. Comparison of chromosome sites labeled with cosmid 16432 showed the distribution of signals on chromosome 1q arm is the same for humans and chimp, but different in the gorilla. Humans and chimps show distinct labeling on sites 1q terminus and 1q41-42. In gorilla, there is instead a large cluster of intense signal near the terminus of 1q that clearly does not extend all the way to the terminus. A paracentric inversion or an unequal cross-over event may account for the observed difference between these species.

  7. Localization of the human transaldolase gene (TALDO) to chromosome 1p33-p34.1 by fluorescence in situ hybridization and PCR analysis of somatic cell hybrids

    SciTech Connect

    Kusuda, Jun; Hashimoto, Katusyuki; Hirai, Momoki

    1997-03-01

    Transaldolase catalyzes the transfer of a C3 fragment corresponding to dihydroxyacetone from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, forming erythrose 4-phosphate and fructose 6-phosphate in the pentose phosphate pathway. The pathway provides mainly D-ribose 5-phosphate for nucleic acid synthesis and NADPH for lipid biosynthesis. 10 refs., 1 fig.

  8. A fluorescence in situ hybridization map of human chromosome 21 consisting of 30 genetic and physical markers on the chromosome: Localization of 137 additional YAC and cosmid clones with respect to this map

    SciTech Connect

    Gingrich, J.C.; Shadravan, F.; Lowry, S.R. )

    1993-07-01

    A fluorescence in situ hybridization (FISH) map of human chromosome 21 was compiled using yeast artificial chromosome (YAC) DNA probes that encode 28 markers physically and/or genetically mapped on the chromosome. Probes that recognize the centromere and rDNA repeat sequences in the p arm were also placed as reference markers on the FISH map. For each probe, the location of the fluorescence hybridization signal was measured on metaphase chromosomes with respect to fractional chromosome length (FL) from p-ter. The location of the markers was established with a standard error of [+-]1.9 Mb using from 9 to 63 FL measurements for each probe. The relative order and separation of the markers as determined by FISH are shown to correspond well to those of other maps of the chromosome. Fifty-one additional YAC and 86 cosmid clones were also localized by FISH with respect to the 30 markers on the chromosome. The cosmids, chosen at random from a flow-sorted chromosome 21 cosmid library, show some biases in chromosome distribution. 42 refs., 2 figs., 3 tabs.

  9. Edited by Oliver Hobert. Last revised May 24, 2012. Published December 13, 2012. This chapter should be cited as: Ji N. and van Oudenaarden A. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos (December 13, 2012),

    E-print Network

    van Oudenaarden, Alexander

    FISH) of C. elegans worms and embryos (December 13, 2012), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.153.1, http://www.wormbook.org. Copyright: © 2012 Ni Ji and Alexander.vanoudenaarden@hubrecht.eu. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos* Ni Ji1

  10. Detection of HEY1-NCOA2 fusion by fluorescence in-situ hybridization in formalin-fixed paraffin-embedded tissues as a possible diagnostic tool for mesenchymal chondrosarcoma.

    PubMed

    Nakayama, Robert; Miura, Yasuhiro; Ogino, Jiro; Susa, Michiro; Watanabe, Itsuo; Horiuchi, Keisuke; Anazawa, Ukei; Toyama, Yoshiaki; Morioka, Hideo; Mukai, Makio; Hasegawa, Tadashi

    2012-12-01

    Mesenchymal chondrosarcoma (MC) is an extremely rare subtype of chondrosarcoma. A tumor specific fusion gene, HEY1-NCOA2 fusion, was recently identified in this tumor. The finding raises the possibility that the diagnosis of MC can be improved by examining the fusion gene. In the present study, we aimed to evaluate the efficacy of fluorescence in situ hybridization (FISH) in detecting HEY1-NCOA2 fusion for the diagnosis of MC. Specimens from 10 patients diagnosed with MC were used for the study. Dual-color FISH was performed using two different probes that specifically hybridize to HEY1 and NCOA2, respectively. Fusion signals were identified in all but two specimens, in which no signal was detected, presumably because of inadequate sample preparation. In accordance with results of a previous study, FISH analysis was highly sensitive in detecting HEY1-NCOA2 fusion in adequately prepared MC samples. The current study adds further support for the use of HEY1-NCOA2 fusion as a valid diagnostic marker for MC. PMID:23252872

  11. Characterization of the temporal persistence of chromosomal abnormalities in the semen of Hodkin`s disease patients after treatment with NOVP chemotherapy using multi-chromosome fluorescence in situ hybridization

    SciTech Connect

    Cassel, M.J.; Robbins, W.A.; Wyrobek, A.J.; Meistrich, M.L.

    1994-12-31

    Three-chromosome fluorescence in situ hybridization (FISH) was applied to sperm of men with Hodgkin`s disease to measure the persistence of chromosomally abnormal sperm within the time interval of 3 to 33 months after the end of treatment. NOVP chemotherapy includes the agents novantrone, oncovin, vinblastine, and prednisone, two of which are spindle poisons expected to induce aneuploidy. Semen samples were evaluated for the frequencies of fluorescence phenotypes representing hyperhaploidy, hypohaploidy, and genomic duplications using DNA probes specific for repetitive sequences on chromosomes X,Y, and 8. Using this procedure, NOVP was previously shown to induce chromosomally abnormal sperm in treated patients. In a longitudinal assessment of 11 semen samples from 2 men, frequencies of abnormal sperm appeared to return to pre-treatment levels at {approximately}6 months after the end of treatment and remained at these levels up to 33 months after the end of treatment. However, pre-treatment frequencies of chromosomally abnormal cells in Hodgkin`s patients were elevated above those found in normal healthy men. Additional patients are being evaluated to determine how long after therapy Hodgkin`s disease patients remain at increased risk for producing chromosomally abnormal sperm.

  12. Whole Mount In Situ Hybridization On Mouse Embryos

    E-print Network

    De Robertis, Eddy M.

    Whole Mount In Situ Hybridization On Mouse Embryos Modified by Lise Zakin and Eddy De Robertis 2008 Henrique et al., 1995. Nature 375, 787-790. Note: This protocol works well for embryos up to 10.5 days post-coïtum (d.p.c.). For older embryos, use in situ on sections. Dissections -Dissect embryos in 1X PBS; remove

  13. Experimental design and quality assurance: in situ fluorescence instrumentation

    USGS Publications Warehouse

    Conmy, Robyn N.; Del Castillo, Carlos E.; Downing, Bryan D.; Chen, Robert F.

    2014-01-01

    Both instrument design and capabilities of fluorescence spectroscopy have greatly advanced over the last several decades. Advancements include solid-state excitation sources, integration of fiber optic technology, highly sensitive multichannel detectors, rapid-scan monochromators, sensitive spectral correction techniques, and improve data manipulation software (Christian et al., 1981, Lochmuller and Saavedra, 1986; Cabniss and Shuman, 1987; Lakowicz, 2006; Hudson et al., 2007). The cumulative effect of these improvements have pushed the limits and expanded the application of fluorescence techniques to numerous scientific research fields. One of the more powerful advancements is the ability to obtain in situ fluorescence measurements of natural waters (Moore, 1994). The development of submersible fluorescence instruments has been made possible by component miniaturization and power reduction including advances in light sources technologies (light-emitting diodes, xenon lamps, ultraviolet [UV] lasers) and the compatible integration of new optical instruments with various sampling platforms (Twardowski et at., 2005 and references therein). The development of robust field sensors skirt the need for cumbersome and or time-consuming filtration techniques, the potential artifacts associated with sample storage, and coarse sampling designs by increasing spatiotemporal resolution (Chen, 1999; Robinson and Glenn, 1999). The ability to obtain rapid, high-quality, highly sensitive measurements over steep gradients has revolutionized investigations of dissolved organic matter (DOM) optical properties, thereby enabling researchers to address novel biogeochemical questions regarding colored or chromophoric DOM (CDOM). This chapter is dedicated to the origin, design, calibration, and use of in situ field fluorometers. It will serve as a review of considerations to be accounted for during the operation of fluorescence field sensors and call attention to areas of concern when making this type of measurement. Attention is also given to ways in which in-water fluorescence measurements have revolutionized biogeochemical studies of CDOM and how those measurements can be used in conjunction with remotely sense satellite data to understand better the biogeochemistry of DOM in aquatic environments.

  14. In situ measurements of phytoplankton fluorescence using low cost electronics.

    PubMed

    Leeuw, Thomas; Boss, Emmanuel S; Wright, Dana L

    2013-01-01

    Chlorophyll a fluorometry has long been used as a method to study phytoplankton in the ocean. In situ fluorometry is used frequently in oceanography to provide depth-resolved estimates of phytoplankton biomass. However, the high price of commercially manufactured in situ fluorometers has made them unavailable to some individuals and institutions. Presented here is an investigation into building an in situ fluorometer using low cost electronics. The goal was to construct an easily reproducible in situ fluorometer from simple and widely available electronic components. The simplicity and modest cost of the sensor makes it valuable to students and professionals alike. Open source sharing of architecture and software will allow students to reconstruct and customize the sensor on a small budget. Research applications that require numerous in situ fluorometers or expendable fluorometers can also benefit from this study. The sensor costs US$150.00 and can be constructed with little to no previous experience. The sensor uses a blue LED to excite chlorophyll a and measures fluorescence using a silicon photodiode. The sensor is controlled by an Arduino microcontroller that also serves as a data logger. PMID:23783738

  15. In situ Measurements of Phytoplankton Fluorescence Using Low Cost Electronics

    PubMed Central

    Leeuw, Thomas; Boss, Emmanuel S.; Wright, Dana L.

    2013-01-01

    Chlorophyll a fluorometry has long been used as a method to study phytoplankton in the ocean. In situ fluorometry is used frequently in oceanography to provide depth-resolved estimates of phytoplankton biomass. However, the high price of commercially manufactured in situ fluorometers has made them unavailable to some individuals and institutions. Presented here is an investigation into building an in situ fluorometer using low cost electronics. The goal was to construct an easily reproducible in situ fluorometer from simple and widely available electronic components. The simplicity and modest cost of the sensor makes it valuable to students and professionals alike. Open source sharing of architecture and software will allow students to reconstruct and customize the sensor on a small budget. Research applications that require numerous in situ fluorometers or expendable fluorometers can also benefit from this study. The sensor costs US$150.00 and can be constructed with little to no previous experience. The sensor uses a blue LED to excite chlorophyll a and measures fluorescence using a silicon photodiode. The sensor is controlled by an Arduino microcontroller that also serves as a data logger. PMID:23783738

  16. Genomic affinities in Turnera (subseries Turnera, Turneraceae) inferred by in situ hybridization techniques.

    PubMed

    López, Alicia; Fernández, Aveliano; Poggio, Lidia

    2010-08-01

    Subseries Turnera comprises a polyploid complex with ploidy levels ranging from diploid (2n = 2x = 10) to octoploid (2n = 8x = 40). The use of fluorescent in situ hybridization greatly improved the knowledge of the karyotypes of Turnera species by detecting and mapping rDNA sites. Interspecific variability in the number of sites was detected, but not in correlation with the ploidy level. A chromosome pair with a strong hybridization signal was always visible and this signal corresponded to the secondary constriction detectable by conventional techniques. Genomic in situ hybridization experiments combined with information on meiotic pairing in species and interspecific hybrids revealed that homologies detected by molecular analysis are greater than those detected by chromosome pairing. This suggests that the formation of the allopolyploids could involve species more closely related than previously assumed. Despite the molecular affinity among the genomes, the meiotic pairing is probably controlled by specific genes that restrict homeologous pairing in polyploids. PMID:20725146

  17. Fluorescence bronchoscopy for localization of carcinoma in situ.

    PubMed

    Profio, A E; Doiron, D R; Balchum, O J; Huth, G C

    1983-01-01

    A fluorescence bronchoscope system has been developed for imaging lung tumors by fluorescence of a previously injected, tumor-specific agent hematoporphyrin derivative. Carcinoma in situ has been localized, but there are too many false positives and negatives. A new system has been implemented which allows rapid switching between viewing of fluorescence, and viewing of the same area under white light illumination as in conventional bronchoscopy. The excitation source is a violet krypton ion laser coupled to a fused quartz fiber light conductor, with a diverging microlens to spread the light uniformly. A third-generation, microchannel plate image intensifier amplifies the weak fluorescence for viewing and video display, recording, and analysis. A movable mirror and periscope bypasses the intensifier for normal color viewing and video display and recording, with the laser shutter closed and the white light shutter open. This facilitates accurate localization, comparison of the color and fluorescence images, and precise sampling during biopsy. The improved system should reduce the false positive rate due to biopsy sampling error, and together with the video analyzer should reduce indeterminate results. PMID:6221184

  18. Small RNA Detection by in Situ Hybridization Methods

    PubMed Central

    Urbanek, Martyna O.; Nawrocka, Anna U.; Krzyzosiak, Wlodzimierz J.

    2015-01-01

    Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics. PMID:26068454

  19. Bayesian fluorescence in situ hybridisation signal classification

    E-print Network

    Lerner, Boaz

    of specific numerical and structural chromosomal abnormalities within these nuclei. Digital microscopy in FISH hybridisation (FISH) signals for the detection of genetic abnormalities. Based on well-discriminating features

  20. A highly specific and sensitive fluorescence in situ hybridization assay for the detection of t(4;11)(q21;q23) and concurrent submicroscopic deletions in acute leukaemias.

    PubMed

    König, Margit; Reichel, Martin; Marschalek, Rolf; Haas, Oskar A; Strehl, Sabine

    2002-03-01

    The translocation t(4;11)(q21;q23) is one of the most frequent 11q23 abnormalities associated with infant leukaemia as well as topoisomerase inhibitor-induced secondary leukaemias. On the molecular level, the MLL gene on 11q23 is fused to the AF4 gene in the 4q21 region, resulting in a chimaeric MLL/AF4 fusion transcript. These particular chromosome rearrangements are generally considered to be associated with poor prognosis, and therefore accurate detection at diagnosis is of clinical significance. In this study we developed a highly specific dual-colour fluorescence in situ hybridization (FISH) assay for the detection of the t(4;11) and demonstrate its usefulness for interphase molecular cytogenetics. In our approach, differentially labelled genomic clones that span the breakpoint cluster regions of both genes involved in the specific translocation were used. Thus, t(4;11)-positive nuclei will display two fusion signals and for t(4;11) cases with concurrent 3' MLL deletions only one fusion signal will be displayed. A very low false-positive value of less than 0.1% was obtained for interphase cells with two fusion signals. In contrast, in cases with 3' MLL deletions that display only one fusion signal, the rate of false-positive nuclei was 10.4%. This FISH assay enables the screening of larger series of patients with haematological diseases for t(4;11) translocations and allows the unambiguous detection of associated cryptic deletions. PMID:11886378

  1. Changes in rRNA Levels during Stress Invalidates Results from mRNA Blotting: Fluorescence In Situ rRNA Hybridization Permits Renormalization for Estimation of Cellular mRNA Levels

    PubMed Central

    Hansen, Martin C.; Nielsen, Allan K.; Molin, Søren; Hammer, Karin; Kilstrup, Mogens

    2001-01-01

    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value. PMID:11466277

  2. Detection of cytokine gene expression in human monocytes and lymphocytes by fluorescent in situ hybridization in cell suspension and flow cytometry.

    PubMed

    Wieckiewicz, J; Krzeszowiak, A; Ruggiero, I; Pituch-Noworolska, A; Zembala, M

    1998-06-01

    The use of digoxigenin (DIG)- and biotin-labelled dsDNA probes to detect TNFalpha-mRNA accumulation in human peripheral blood mononuclear cells (PBMC) and isolated monocytes is described. The fragment of the glyceraldehyde-3-phosphate dehydrogenase GAPDH-cDNA was used as a control probe. The hybridization signals were detected by staining with fluorescein isothiocyanate (FITC)-labelled anti-DIG antibody and avidin-FITC, respectively. The cells were stimulated in vitro with lipopolysaccharide (LPS) for 0.5-6 h. The TNFalpha-mRNA was detected in monocytes 1 h after stimulation with LPS, and the highest accumulation was seen around 2 h. The TNFalpha-mRNA in stimulated PBMC was detected at the lower level peaking around 4 h. The TNFalpha-mRNA accumulation was lower in lymphocytes than in monocytes when PBMC were studied. There was no difference in the level of GAPDH-mRNA between unstimulated and stimulated cells. Finally, an enhanced accumulation of TNFalpha-mRNA was observed in PBMC from some patients with sepsis or cancer. Thus, this study shows that cytokine gene expression may be detected in cells ex vivo. This opens the possibility of studying the level of cytokine gene activation in PBMC of patients with diseases where the role of cytokines in their pathophysiology is implicated. PMID:9852637

  3. A new classification of interphase nuclei based on spatial organizations of chromosome 8 and 21 for t(8;21) (q22;q22) acute myeloid leukemia by three-dimensional fluorescence in situ hybridization.

    PubMed

    Tian, Xueli; Wang, Yanfang; Zhao, Fengying; Liu, Jinlin; Yin, Jun; Chen, Dieyan; Ma, Wanyun; Ke, Xiaoyan

    2015-12-01

    Interphase heterogenous chromosomes spatially close to each other are predominantly located near the center of nuclei and are prone to incur translocations. We screened a t(8;21) (q22;q22) acute myeloid leukemia-M2 patient during three phases (post-chemotherapy, remittent stage, and relapse) and a donor of normal karyotype as control by two-(2D) and three-dimensional (3D)-fluorescence in situ hybridization (FISH). Our classification of nuclei (normal, transitional, and malignant nuclei) by 3D-FISH analyses may provide a more precise prognosis than 2D-FISH results, especially for remittent stage sample in our study, in which 2D-FISH findings showed normal results, whereas 3D-FISH results showed extreme abnormalities (normal nuclei 27%, transitional nuclei 36%, malignant nuclei 37%). The relative radial positions (d/R) of chromosomes 8 were similar to d/R of chromosomes 21 for the relapse sample. We classified heterogenous chromosome pairs into close pairs and normal pairs based on their relative distances (d'/(2R)). The centers of close pairs were more internal than normal pairs in nuclei in all samples, and the d/R values of a given-type pairwise heterogenous chromosomes were similar among four samples. Our data demonstrate that the classification of nuclei based on spatial organization of chromosomes by 3D-FISH is reasonable and essential for evaluating acute myeloid leukemia prognosis. PMID:26423235

  4. Determination of HER2 and p53 Mutations by Sequence Analysis Method and EGFR/Chromosome 7 Gene Status by Fluorescence in Situ Hybridization for the Predilection of Targeted Therapy Modalities in Immunohistochemically Triple Negative Breast Carcinomas in Turkish Population.

    PubMed

    Pala, Emel Ebru; Bayol, Umit; Keskin, Elif Usturali; Ozguzer, Alp; Kucuk, Ulku; Ozer, Ozge; Koc, Altug

    2015-09-01

    Triple negative breast cancer (TNBC), an agressive subtype accounts nearly 15 % of all breast carcinomas. Conventional chemotherapy is the only treatment modality thus new, effective targeted therapy methods have been investigated. Epidermal growth factor receptor (EGFR) inhibitors give hope according to the recent studies results. Also therapeutic agents have been tried against aberrant p53 signal activity as TNBC show high p53 mutation rates. Our aim was to detect the incidence of mutations/amplifications identified in TNBC in our population. Here we used sequence analysis to detect HER2 (exon 18-23), p53 (exon 5-8) mutations; fluorescence in situ hybridization (FISH) method to analyse EGFR/chromosome 7 centromere gene status in 82 immunohistochemically TNBC. Basaloid phenotype was identified in 49 (59.8 %) patients. EGFR amplification was noted in 5 cases (6.1 %). All EGFR amplified cases showed EGFR overexpression by immunohistochemistry (IHC). p53 mutations were identified in 33 (40.2 %) cases. Almost 60 % of the basal like breast cancer cases showed p53 mutation. Only one case showed HER2 mutation (exon 20:g.36830_3). Our results showed that gene amplification is not the unique mechanism in EGFR overexpression. IHC might be used in the decision of anti-EGFR therapy in routine practice. p53 mutation rate was lower than the rates reported in the literature probably due to ethnic differences and low sensitivity of sanger sequences in general mutation screening. We also established the rarity of HER2 mutation in TNBC. In conclusion EGFR and p53 are the major targets in TNBC also for our population. PMID:26060045

  5. Interstitial deletion of chromosome 1q [del(1)(q24q25.3)] identified by fluorescence in situ hybridization and gene dosage analysis of apolipoprotein A-II, coagulation factor V, and antithrombin III

    SciTech Connect

    Takano, Takako; Yamanouchi, Yasuko; Mori, Yosuke

    1997-01-20

    We report on a 12-month-old Japanese boy with an interstitial deletion of the long-arm of chromosome 1 and meningomyelocele, hydrocephalus, anal atresia, atrial septal defect, left renal agenesis, bilateral cryptorchidism, talipes equinovarus, low birth weight, growth/developmental retardation, and many minor anomalies. By conventional GTG-banding, his karyotype was first interpreted as 46,XY,de1(1)(q23q24), but it was corrected as 46,XY.ish del(1)(q24q25.3) by fluorescence in situ hybridization using 11 known cosmid clones as probes. His serum levels of apolipoprotein A-II (gene symbol: APOA2, previously assigned to 1q21-q23) and coagulation factor V (F5, 1q21-q25) were normal, while serum concentration and activity of antithrombin III (AT3, 1q23-q25.1) was low. The results indicated that localization of APOA2 and F5 are proximal to the deleted region and AT3 is located within the deletion extent in the patient. 16 refs., 4 figs.

  6. Detection of chromosome aneuploidy in breast lesions with fluorescence in situ hybridization: Comparison of whole nuclei to thin tissue sections and correlation with flow cytometric DNA analysis

    SciTech Connect

    Visscher, D.W.; Wallis, T.; Ritchie, C.A.

    1995-09-01

    We compared flow-cytometric DNA histogram pattern to counts of 4 fluorescent-labelled centromeric probes (chromosomes 1, 7, 8, and 17) in whole nuclei (WN) and in nuclei from formalin-fixed deparaffinized thin tissue section (TS) in 25 breast lesions. In benign lesions, signal gains (i.e., trisomic nuclei) were never observed in greater than 10% of nuclei from either WN or TS preparations. Loss of signal in benign breast lesions, however, varied considerably (0-43%) between individual case and between chromosome probes. The mean incidence of signal loss in WN of benign lesions ranged from 8.9% (chromosome 7) to 14.4 % (chromosome 1) of nuclei. These signal loss frequencies exceeded those of benign lymphoid control cells. In three benign lesions, signal loss in WN (with one probe) was observed in at least 25% of nuclei. Signal losses in benign TS, on average, were 50-150% greater than in matched WN preparations (chromosome 1: 21.7%, chromosome 7: 21.5%). Malignant lesions generally, but not always, displayed fewer monosomic nuclei and more trisomic nuclei in compared to TS, compatible with a slicing (i.e., nuclear truncation) artifact. Signal counts in carcinomas correlated well with flow cytometric DNA index; however, they were also characterized by evidence of genetic instability, manifest as signal gains in a subset of nuclei (10-25%) with individual probes in diploid range cases, as well as intratumoral heterogeneity, reflected as discrepancies in probe counts between WN and TS samples. We conclude that signal losses with centromeric probes are largely, but not entirely, explained by nuclear slicing. The minimum signal loss threshold for establishment of monosomy using interphase cytogenetics is thus unclear, even in WN. Signal gains indicative of trisomy, in contrast, are reliably associated with malignancy and may reflect gross DNA aneuploidy as well as genetic instability. 10 refs., 1 fig., 3 tabs.

  7. Fixing Embryos for In Situ Hybridization Leslie Vosshall

    E-print Network

    1 Fixing Embryos for In Situ Hybridization Leslie Vosshall 2/7/2001 1. A few days in advance, set old plates in the fly waste container in the fly room. 3. The evening before the first embryo the embryos on the old plate as described below. First, use a spatula or kimwipe to remove the large blob

  8. Applications of Strand-Specific in situ Hybridization

    SciTech Connect

    Goodwin, E.H.; Meyne, J.; Bailey, S.M.; Quigley, D.; Smith, L.; Tennyson, R.

    1997-01-01

    This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Fluorescence in situ hybridization (FISH) is used to determine the location of specific DNA sequences on chromosomes. It is an effective tool in genomic mapping and is finding increasing use in medical diagnosis. A ''strand-specific'' version of FISH has been developed in the Life Sciences Division of LANL. The new procedure, named CO-FISH, reveals not only location but also the 5'-to-3'direction of a target sequence, such as the sense strand of a gene. This project was designed to investigate applications of the new technique. Strand-specific FISH was found to be useful and informative for genomic mapping of repetitive DNA sequences. The method provide a valuable new tool for investigating the mechanisms of aneuploidy inducing agents and the cytogenetic phenomena called lateral asymmetry. Finally, using strand-specific FISH, the authors were able to detect certain types of chromosome aberrations (isochromosomes, inversions and Robertsonian translocations) that can be difficult to observe with standard techniques.

  9. Chromosome mapping of 18S rDNA and 5S rDNA by dual-color fluorescence in situ hybridization in the half-smooth tongue sole (Cynoglossus semilaevis).

    PubMed

    Jiang, L; Jiang, J; Liu, J; Yuan, J; Chen, Y; Zhang, Q; Wang, X

    2014-01-01

    Half-smooth tongue sole (Cynoglossus semilaevis) is an important aquaculture flatfish in China. Cytogenetic analysis has revealed that its sex determination system is female heterogametic (ZZ/ZW). The W chromosome is morphologically larger and has been considered evolutionarily younger than any other chromosome in the set. However, the genetic origin and evolution process of this neo-chromosome remains unclear. In this study, 2 tandem arrays of rRNA genes were chosen to address this question. Both the major rDNA (18S rDNA) and the minor rDNA (5S rDNA) were located on the C. semilaevis chromosomes by fluorescence in situ hybridization (FISH). Six 18S rDNA signals were observed on the centromeric regions of 3 pairs of autosomes in both males and females. In females, there was an additional 18S rDNA signal mapping to the telomeric region of the W chromosome long arm. With respect to the 5S rDNA, 12 signals were mapped to the centromeric regions of six pairs of autosomes. Two-color FISH further confirmed that the two pairs of the 5S rDNA signals were correspondingly located at the same positions of the same autosomes as those of the 18S rDNA signals. These results allowed us to speculate about the evolution process of the W chromosome. Chromosome fusions and repetitive sequence accumulations might have occurred in C. semilaevis. The synteny and non-synteny of C. semilaevis 18S rDNA and 5S rDNA might imply the original and evolutionary characteristics of this species. These findings will facilitate studies on karyotype evolution of the order Pleuronectiformes. PMID:25526196

  10. Identification of dermatophyte species using genomic in situ hybridization (GISH).

    PubMed

    Worek, Mariusz; Kwiatkowska, Aleksandra; Ciesielska, Anita; Jaworski, Adam; Kaplan, Jakub; Miedziak, Beata; Deregowska, Anna; Lewinska, Anna; Wnuk, Maciej

    2014-05-01

    A genomic in situ hybridization (GISH)-based method for dermatophyte identification has been developed. Using specific GISH probes, discrimination between Trichophyton interdigitale, Trichophyton rubrum and Microsporum canis has been conducted. Moreover, GISH has been found particularly helpful when proper dermatophyte identification was difficult due to ambiguous PCR-RFLP patterns. PMID:24589627

  11. Nonradioactive In Situ Hybridization on Skeletal Tissue Sections

    E-print Network

    indicate the loca- tion of the transcript. Key words In situ hybridization, Digoxigenin, Biotin, biotin, and fluorescein, to nucleotides. This innovation has transformed the technique into a fast or fluorescein or biotin labeling mix (Roche). 3. Transcription buffer (Roche). 4. Ribonuclease (RN

  12. Assessment of HER2 Status Using Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) Techniques in Mucinous Epithelial Ovarian Cancer: A Comprehensive Comparison between ToGA Biopsy Method and ToGA Surgical Specimen Method

    PubMed Central

    Chao, Wan-Ru; Lee, Ming-Yung; Ruan, Alexandra; Sheng, Huang Pin; Hsu, Jeng-Dong; Han, Chih-Ping; Koo, Chiew-Loon

    2015-01-01

    We aimed to compare the assay performance characteristics of HER2 status in mucinous epithelial ovarian cancer (EOC) by ToGA (Trastuzumab for Gastric Cancer) biopsy versus ToGA surgical specimen methods. Forty-nine tissue microarray (TMA) samples of mucinous EOC from Asian women were analyzed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) tests using ToGA trial HER2 scoring methods. The overall concordance between IHC and FISH by the ToGA surgical specimen method is 97.56% and by the ToGA biopsy specimen method is 97.14%. The agreements of HER2 IHC results under both biopsy and surgical specimen methods were nearly perfect (weighted kappa = 0.845). Additionally, the percentage of Her2 FISH amplification showed increasing trend with increasing HER2 IHC ordinals (negative, equivocal, positive) by both TOGA biopsy (P<0.001) and surgical specimen method (P<0.001). After excluding equivocal cases, the sensitivity (100%), PPV (88.89%) and NPV (100%) of HER2 IHC were unchanged under either surgical specimen method or biopsy method. However, the specificity (96.97%) and accuracy (97.56%) of HER2 IHC was slightly higher under the surgical specimen method than those (specificity 96.30%, accuracy 97.14%) under the biopsy method. Of the total 49 cases, the number (n = 14) of HER2 IHC equivocal results under the ToGA biopsy method was 1.75-fold higher than those (n = 8) under the ToGA surgical specimen method (28.57% vs. 16.32%). Therefore, compared to ToGA surgery specimen method, the ToGA biopsy method caused more equivocal IHC cases to be referred to FISH testing and did not increase the detection rates of Her2 FISH amplification. PMID:26566289

  13. Fluorescence in situ hybridization (FISH) mapping of human chromosome 1: Cytogenetic band localization of 71 NotI linking clones on chromosome 1q25

    SciTech Connect

    Sumegi, J.; Talmadge, C.G.; Zhen, D.K.

    1994-09-01

    Seventy-one human chromosome 1q25-qter-specific lambda clones have been isolated from NotI-linking libraries which were constructed using DNA from MCH206.1 somatic cell hybrid cells. These cells contain chromosome 1q25 translocated to chromosome Xp22 as the only human chromosomes in a mouse background. The NotI-linking clones have been mapped to cytogenetic bands. The relative order of ten NotI clones in 1q32 and 1q41 and their relation to known chromosome 1 markers have been also determined. Portions of these ten NotI-linking clones were sequenced. Most of the NotI-linking clones were derived from CpG islands, which are often associated with genes. DNA sequence homologies were searched for these ten NotI-linking clones in sequences available in GeneBank. One of the NotI clones carries sequences identical to TGF-beta 2. The NotI-linking clones described here will be useful for constructing a long-range restriction map of chromosome 1q25-qter and may contribute to the cloning of disease genes.

  14. The fate of Helicobacter pylori phagocytized by Acanthamoeba polyphaga demonstrated by fluorescent in situ hybridization and quantitative polymerization chain reaction tests

    EPA Science Inventory

    Helicobacter pylori able to express green fluorescent protein, as well as an ATCC strain, and a clinical isolate of this pathogen were evaluated for their ability to survive predation by Acanthamoeba polyphaga. Ingestion was evaluated by microscopic observation of the GFP-H. pyl...

  15. Combined microRNA in situ hybridization and immunohistochemical detection of protein markers.

    PubMed

    Nielsen, Boye Schnack; Holmstrøm, Kim

    2013-01-01

    MicroRNAs are short (18-23 nucleotides) non-coding RNAs involved in posttranscriptional regulation of gene expression through their specific binding to the 3'UTR of mRNAs. MicroRNAs can be detected in tissues using specific locked nucleic acid (LNA)-enhanced probes. The characterization of microRNA expression in tissues by in situ detection is often crucial following a microRNA biomarker discovery phase in order to validate the candidate microRNA biomarker and allow better interpretation of its molecular functions and derived cellular interactions. The in situ hybridization data provides information about contextual distribution and cellular origin of the microRNA. By combining microRNA in situ hybridization with immunohistochemical staining of protein markers, it is possible to precisely characterize the microRNA expressing cells and to identify the potential microRNA targets. This combined technology can also help to monitor changes in the level of potential microRNA targets in a therapeutic setting. In this chapter we present a fluorescence-based technology that allows the combination of microRNA in situ hybridization with immunohistochemistry exemplified by the in situ detection of miR-21 and miR-205 in combination with PDCD4 and smooth muscle a-actin. PMID:23436423

  16. The detection of HIV by in situ hybridization

    SciTech Connect

    Shapshak, P.; Sun, N.C.; Resnick, L.; Hsu, M.Y.; Tourtellotte, W.W.; Schmid, P.; Conrad, A.; Fiala, M.; Imagawa, D.T. )

    1990-03-01

    A simplified method of in situ hybridization is described for the detection of HIV targets. This standardized method can be applied to sections of formalin-fixed paraffin-embedded tissues, cell blocks, and smears of cultured cells using {sup 3}H- or {sup 35}S-labeled DNA and {sup 35}S-labeled RNA probes. In order to use elevated stringencies in the hybridization and wash steps, tissue sections and cells are covalently bonded to silanated glass slides without their subsequent loss from the slides. Routine hematoxylin and eosin or Romanovsky's stains enable the identification of the cells detected by in situ hybridization. HIV-infected neuroblastoma and lymphoid cell lines, lymph nodes, bone marrow, kidney, as well as brain tissue from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex are used to demonstrate the generality of the procedure. The standardized method described widens the ease and applicability of in situ hybridization in the investigation of pathologic tissues with the use of diverse specimens and probes.

  17. DETECTION OF HYPERDIPLOIDY IN RAT INTERPHASE HEPATOCYTES FOLLOWING TREATMENT IN VIVO WITH VINBLASTINE SULFATE USING FLUORESCENCE IN SITU HYBRIDIZATION (FISH). (R826408)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  18. ENHANCING THE IN VITRO AND IN VIVO DETECTION OF ANEUPLOIDY BY FLUORESCENCE IN SITU HYBRIDIZATION WITH THE USE OF BROMODEOXYURIDINE AS A PROLIFERATION MARKER. (R826408)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  19. In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods.

    PubMed Central

    Kim, B; Chae, C

    2001-01-01

    Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available. Images Figure 1. Figure 2. PMID:11227192

  20. Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

    PubMed

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2015-02-01

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on paraffin-embedded material to test for other diagnostically, prognostically, or therapeutically relevant genomic mutations in lipomatous tumors. PMID:25679065

  1. Detection of MDM2/CDK4 Amplification in Lipomatous Soft Tissue Tumors From Formalin-fixed, Paraffin-embedded Tissue: Comparison of Multiplex Ligation-dependent Probe Amplification (MLPA) and Fluorescence In Situ Hybridization (FISH).

    PubMed

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2014-01-30

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on paraffin-embedded material to test for other diagnostically, prognostically, or therapeutically relevant genomic mutations in lipomatous tumors. PMID:24487315

  2. DETECTION OF HYPERDIPLOIDY IN BLADDER EPITHELIAL CELLS OF RATS TREATED WITH ORTHO-PHENYLPHENOL USING FLUORESCENCE IN SITU HYBRIDIZATION. (R826408)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  3. Apparent mosaicism for del(17)(p11.2) ruled out by fluorescence in situ hybridization in a Smith-Magenis syndrome patient

    SciTech Connect

    Juyal, R.C.; Shaffer, L.G.; Lupski, J.R.; Greenberg, F.; Baldini, A.; Patel, P.I.

    1995-11-20

    Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome typically associated with a deletion of band p11.2 of human chromosome 17. Finucane et al. reported a 14-year-old boy with mild physical and behavior manifestations of SMS. No evidence for deletion was initially evident in 20 peripheral blood lymphocytes examined at 850 band level of resolution. Examination of metaphase chromosomes of skin fibroblasts showed a deletion of 17p11.2 in 25/25 cells examined which was consistent with the patient`s clinical manifestations of SMS. Subsequent examination of 25 cells from peripheral blood cultures indicated that 11% of cells harbored a deletion at 17p11.2, thus suggesting a mosaicism for the deletion. A third study of 20 peripheral blood lymphocytes examined at 550-850 band length resolution in a different laboratory, indicated that 13 cells had no apparent deletion, 4 cells had an apparent deletion and 3 cells were questionable. 7 refs.

  4. Evaluation of Dual-Color Fluorescence In Situ Hybridization With Peptide Nucleic Acid Probes for the Detection of Mycobacterium tuberculosis and Non-Tuberculous Mycobacteria in Clinical Specimens

    PubMed Central

    Kim, Namhee; Lee, Seung Hee; Yi, Jongyoun

    2015-01-01

    Background Peptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes. Methods PNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain. Results The MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676). Conclusions Dual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered. PMID:26206686

  5. International, collaborative assessment of limitations of chromosome-specific probes (CSP) and fluorescent in situ hybridization (FISH): Analysis of expected detections in 73,000 prenatal cases

    SciTech Connect

    Evans, M.I.; Henry, G.P.; Miller, W.A.

    1994-09-01

    FISH and CSP have been proposed to reduce karyotyping need. The purpose of this study was to assess the potential efficacy of CSP-FISH using currently available probes (13, 18, 21, X, & Y) in large, prenatal diagnostic centers. Results (1990-1993) from 7 centers in 4 countries were divided by those expected to be detectable by currently available probes, and those which would be missed assuming 10% probe efficacy. 72,994 karyotypes included 699 trisomy 21`s, 352 trisomy 18`s, 136 trisomy 13`s, 358 sex chromosome aneuploidies, 70 triploidies, and 855 others (translocations, inversions, deletions, markers). Of 2,613 abnormalities, 1,745 would be detectable (66.8%). [Detroit 55.7%, Stockholm 68.3%, Boston 52.6%, Denver 61.3%, Muenster 77.0%, London 84.5%, Philadelphia 69.4%]. Centers with high proportions of referrals for ultrasound anomalies had the highest CSP-FISH positives secondary to increased T 18 & 13. We conclude: (1) 73,000 karyotypes show relatively consistent incidences of the common trisomies, sex chromosome abnormalities, and other chromosome abnormalities among the centers. (2) The proportion expected detectable by FISH-CSP technology varies from 52.6% to 84.5%, averaging 66.8%. (3) 1/3 of the karyotypic abnormalities would be missed, and therefore, replacement of complete karyotyping with FISH would have unacceptably high false-negative rates for routine evaluation. (4) FISH-CSP, while useful when positive for anomalies, is not sufficient when negative to obviate the need for a complete karyotype.

  6. Detection of numerical alterations for chromosomes 7 and 12 in benign thyroid lesions by in situ hybridization. Histological implications.

    PubMed Central

    Criado, B.; Barros, A.; Suijkerbuijk, R. F.; Weghuis, D. O.; Seruca, R.; Fonseca, E.; Castedo, S.

    1995-01-01

    Polysomies of chromosomes 7 and 12 have been frequently observed by conventional cytogenetics in a subgroup of thyroid follicular adenomas and in some cases of thyroid goiters. To further study possible cytogenetic similarities between these two types of thyroid lesions, we have used fluorescence in situ hybridization (FISH) to detect polysomies of chromosomes 7 and/or 12 in isolated nuclei from frozen and paraffin-embedded material of goiters and thyroid follicular adenomas and compared results with previous ones obtained by flow cytometry and conventional cytogenetics. With a set of two alpha-satellite DNA probes specific for the centromeric regions of chromosomes 7 and 12, used either separately (single-target fluorescence in situ hybridization) or simultaneously (double-target fluorescence in situ hybridization), we detected polysomies of chromosome 7 in 35.7% of the thyroid follicular adenomas and in 10.7% of the goiters. Polysomies of chromosome 12 were detected in 29.6% of the thyroid follicular adenomas and 6.7% of the goiters. The significantly higher frequency of adenomas with numerical alterations for chromosomes 7 and/or 12 supports the idea of a biological continuum and karyotypic evolution between both lesions. It is also noteworthy that polysomies of chromosomes 7 and/or 12 were observed only in lesions with an exclusive (or predominant) microfollicular histological component, as detected by enzymatic in situ hybridization on frozen sections. Images Figure 1 Figure 2 Figure 3 PMID:7604875

  7. In-situ hydrocarbon delineation using laser-induced fluorescence

    SciTech Connect

    Taer, A.D.; Hastings, R.W.; Brown, A.Y.; Frend, R.

    1996-12-01

    An investigation of hydrocarbons in soils was conducted at an active Shell Oil Company petroleum products terminal, located in Carson, California. An investigation approach involving Laser-Induced Fluorescence (LIF) and Cone Penetrometer Testing (CPT) technologies was implemented to provide real-time, in-situ characterization of site stratigraphy, hydrocarbon distribution and importantly, hydrocarbon product differentiation. The area of investigation is located along a property boundary, where a plume of separate phase hydrocarbons has been actively recovered for several years. CPT/LIF technology was selected for the investigation since previous delineation efforts using hydrocarbon fingerprinting methods proved inconclusive. Additionally, the CPT/LIF technology had the potential to provide a cost effective solution to accomplish project objectives. Based on the information obtained during this investigation, it was determined that the plume of separate phase hydrocarbons along the northern property boundary is from a source distinctly different than any identified hydrocarbons known to be from on-site sources. In addition, the plume was determined to not be connected with any other known on-site hydrocarbon plumes. The results of this CPT/LIF investigation were consistent with the known hydrogeologic conditions. This evaluation determined that CPT/LIF technology was very effective in addressing project objectives and resulted in a significant cost savings.

  8. Enumeration of semen leucocytes by fluorescence in situ hybridisation technique.

    PubMed

    Conte, R A; Luke, S; Verma, R S

    1995-12-01

    Aim-To determine whether the fluorescent in situ hybridisation technique (FISH) using a total human DNA genomic probe can be used to enumerate semen leucocytes.Methods-Semen samples from five donors were subjected to a mild KC1 solution. These samples were then biotin labelled under FISH conditions using a total human DNA genomic probe and the leucocyte counts were determined. To check the accuracy of the technique a monoclonal antibody against the common leucocyte antigen CD45 [KC56(T-200)] served as a control. An isotypic control for [KC56(T-200)], the immunoglobulin [MsIgG1], served as a secondary control.Results-Semen leucocytes stained by the FISH technique were easily detected because of their distinct bright yellow colour, while the sperm cells were red. The leucocyte count ranged from 0.5 to 4.9 x 10(6) per ml of semen. KC56(T-200) and its isotypic control MsIgG1, which served as control for the FISH technique, accurately identified 94% and 97% of the semen leucocytes of a control donor, respectively.Conclusions-The FISH technique using a total human DNA probe can accurately and effectively enumerate the overall leucocyte population in semen. PMID:16696031

  9. Immunohistochemistry and RNA in situ hybridization in mouse brain development.

    PubMed

    Liu, Jinling; Liu, Aimin

    2014-01-01

    During development, the mouse brain is progressively divided into functionally distinct compartments. Numerous neuronal and glial cell types are subsequently generated in response to various inductive signals. Each cell expresses a unique combination of genes encoding proteins from transcription factors to neurotransmitters that define its role in brain function. To understand these important and highly sophisticated processes, it is critical to accurately locate the various proteins and cells that produce them. In this chapter, we introduce the techniques of immunohistochemistry, which detects the localization of specific proteins, and RNA in situ hybridization, which enables the visualization of specific mRNAs. PMID:24048940

  10. Localization of ARHG, a member of the RAS homolog gene family, to 11p15. 5-11p15. 4 by fluorescence in situ hybridization

    SciTech Connect

    Taviaux, S.A.; Demaille, J.G. ); Vincent, S.; Fort, P. )

    1993-06-01

    The RAS superfamily proteins are GTP-binding proteins that act in the pathway of signal transduction and play a key role in the regulation of cellular functions. For example, the mammalian RAS gene products are 21-kDa proteins that control regulatory pathways critical for normal proliferation and differentiation. Specific mutations in RAS-related genes alter the gene product and may be involved in mechanisms that promote neoplastic transformation in human cells. The RAS superfamily can be divided into four groups. One of them is the RAS homolog gene family, a new member of which, ARHG, has been isolated from hamster and human. The ARHG gene is a late induced gene, and RNA accumulation is proportional to the strength of the mitogen used. Phylogenetic studies suggest that it might have diverged early during evolution. 11 refs., 1 fig.

  11. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

    PubMed Central

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis. PMID:26305575

  12. The application of spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) technology to determine the chromosomal content(s) of micronuclei.

    PubMed

    Leach, N T; Jackson-Cook, C

    2001-08-22

    DNA loss by the process of micronucleation is associated with aging, cancer and environmental exposure. The primary aim of this study was to identify the chromosomal origin of the DNA excluded into micronuclei (MN). This was achieved using a novel application of SKY and FISH technologies. Cytochalasin B (Cyt B)-treated lymphocyte cultures from three females (aged 28, 42 and 72) were analyzed. SKY revealed that the majority of MN (89.8, 82.9, and 97.6% in the 28-, 42- and 72-year-old (y.o.), respectively) had a uniform, single color, suggesting that they were comprised of DNA from a single chromosome. Using a pancentromeric probe, most of the MN (82% in 28 y.o., 69% in 42 y.o. and 80% in 72 y.o.) had one centromere signal present. Overall, the confirmation studies (using FISH with chromosome-specific WCP) were in agreement with the SKY chromosomal assignments for 71.1% of the MN. Although the SKY analysis showed that all of the 23 chromosomes (22 autosomes and the X chromosome) could be present in the MN, overall, the X chromosome was seen most frequently. DNA from the X chromosome was seen in 50.6% of MN in the 42 y.o. individual, whereas in the 28 and 72 y.o. it was seen in 12.2 and 7.1% of MN, respectively. This difference (P<0.0001) in the frequencies of X chromosome exclusion into MN among individuals was independently confirmed using a single whole chromosome painting probe (WCP) for the X chromosome. SKY also showed variation in the frequency of autosomal exclusion into MN between chromosomes and between females. Collectively, this study supports the hypothesis that the majority of MN contain DNA from a single, monocentric chromosome. The use of SKY technology for the identification of the chromosomal content(s) of MN provides an opportunity for expansion of our knowledge of the chromosomal changes that accompany MN formation. PMID:11448638

  13. Localization of the human UGP2 gene encoding the muscle isoform of UDPglucose pyrophosphorylase to 2p13-p14 by fluorescence in situ hybridization

    SciTech Connect

    Cheng, Sou-De; Peng, Hwei-Ling; Chang, Hwan-You

    1997-02-01

    UDPglucose pyrophosphorylase (UGP, EC 2.7.7.9) catalyzes the transfer of a glucose moiety from Glc1P to MgUTP, forming UDPglc and MgPPi. This reaction is necessary in several tissues. In liver and muscle, UDPglc is the direct precursor of glycogen, while in lactating mammary gland it is converted to UDPgalactose and thence lactose. Liver also requires UDPglc for the formation of UDPglucuronate, which then acts as a source for the formation of soluble glucuronides of xenobiotic and endobiotic metabolites destined for excretion. 6 refs., 1 fig.

  14. Molecular Diversity, Cultivation, and Improved Detection by Fluorescent In Situ Hybridization of a Dominant Group of Human Gut Bacteria Related to Roseburia spp. or Eubacterium rectale

    PubMed Central

    Aminov, Rustam I.; Walker, Alan W.; Duncan, Sylvia H.; Harmsen, Hermie J. M.; Welling, Gjalt W.; Flint, Harry J.

    2006-01-01

    Phylogenetic analysis was used to compare 16S rRNA sequences from 19 cultured human gut strains of Roseburia and Eubacterium rectale with 356 related sequences derived from clone libraries. The cultured strains were found to represent five of the six phylotypes identified. A new oligonucleotide probe, Rrec584, and the previous group probe Rint623, when used in conjunction with a new helper oligonucleotide, each recognized an average of 7% of bacteria detected by the eubacterial probe Eub338 in feces from 10 healthy volunteers. Most of the diversity within this important group of butyrate-producing gut bacteria can apparently be retrieved through cultivation. PMID:16957265

  15. Delay to formalin fixation 'cold ischemia time': effect on ERBB2 detection by in-situ hybridization and immunohistochemistry.

    PubMed

    Portier, Bryce P; Wang, Zhen; Downs-Kelly, Erinn; Rowe, Jordi J; Patil, Deepa; Lanigan, Chis; Budd, G Thomas; Hicks, David G; Rimm, David L; Tubbs, Raymond R

    2013-01-01

    The American Society of Clinical Oncology/College of American Pathologists ERBB2 testing guidelines address several pre-analytical variables known to affect ERBB2 testing accuracy. According to 2010 updated guidelines, the pre-analytical variable of time to tissue fixation (cold ischemia time) should be kept to <1?h, however, little has been published about cold ischemia time and its significance in ERBB2 testing. To that end, this study evaluated ERBB2 status using two different FDA-approved in-situ hybridization methods and an FDA-approved immunohistochemistry (IHC) assay in the largest cohort to date (n=84) of invasive breast carcinomas with tracked cold ischemia time. Cold ischemia time was stratified into four groups (<1?h (n=45), 1-2?h (n=27), 2-3?h (n=6), and >3?h (n=6)) and ERBB2 status was evaluated in each group by IHC (4B5) and by in-situ hybridization methodologies (PathVysion(®) fluorescence in situ hybridization and the INFORM HER2(®) dual in situ DNA probe assay). Both in-situ hybridization methods were evaluated using three ERBB2 scoring criteria (dual-probe guidelines, single-probe guidelines, and the FDA package insert scoring instructions). Fluorescence in-situ hybridization (FISH) and INFORM HER2(®) demonstrated 100% concordance in the detection of ERBB2 amplification by all three scoring guidelines at all cold ischemia time points. Agreement between in-situ hybridization methodologies and IHC was superior using single-probe guidelines compared with dual probe or FDA scoring instructions. In addition, Inform HER2(®) in-situ hybridization signals were significantly more intense than FISH at all cold ischemia time points, however, no significant loss of either chromosome 17 or ERBB2 signal was detected by FISH or Inform HER2(®) in-situ hybridization in cold ischemia times up to 3?h. On the basis of our findings, cold ischemia time up to 3?h has no deleterious effect on the detection of ERBB2 via in-situ hybridization or IHC. PMID:22899285

  16. In situ measurements of colloid transport and retention using synchrotron X-ray fluorescence

    E-print Network

    In situ measurements of colloid transport and retention using synchrotron X-ray fluorescence David] The physics regarding the retention and mobilization of colloids in saturated and unsaturated conditions remains poorly understood, partially because of the inability to measure colloid concentrations in situ

  17. Root genomics: towards digital in situ hybridization

    PubMed Central

    Scheres, Ben; van den Toorn, Henk; Heidstra, Renze

    2004-01-01

    Separation of cell types and developmental stages in the Arabidopsis root and subsequent expression profiling have yielded a valuable dataset that can be used to select candidate genes for detailed study and to start probing the complexities of gene regulation in plant development. PMID:15186485

  18. Hybrid-type temperature sensor for in situ measurement

    SciTech Connect

    Iuchi, Tohru; Hiraka, Kensuke

    2006-11-15

    A hybrid-type surface temperature sensor combines the contact and noncontact methods, which allows us to overcome the shortcomings of both methods. The hybrid-type surface thermometer is composed mainly of two components: a metal film sheet that makes contact with an object and a radiometer that is used to detect the radiance of the rear surface of the metal film, which is actually a modified radiation thermometer. Temperature measurement using the hybrid-type thermometer with a several tens micrometer thick Hastelloy sheet, a highly heat and corrosion resistant alloy, is possible with a systematic error of -0.5 K and random errors of {+-}0.5 K, in the temperature range from 900 to 1000 K. This thermometer provides a useful means for calibration of in situ temperature measurement in various processes, especially in the silicon semiconductor industry. This article introduces the basic idea of the hybrid-type surface sensor, presents experimental results and discussions, and finally describes some applications.

  19. Formaldehyde-based whole-mount in situ hybridization method for planarians.

    PubMed

    Pearson, Bret J; Eisenhoffer, George T; Gurley, Kyle A; Rink, Jochen C; Miller, Diane E; Sánchez Alvarado, Alejandro

    2009-02-01

    Whole-mount in situ hybridization (WISH) is a powerful tool for visualizing gene expression patterns in specific cell and tissue types. Each model organism presents its own unique set of challenges for achieving robust and reproducible staining with cellular resolution. Here, we describe a formaldehyde-based WISH method for the freshwater planarian Schmidtea mediterranea developed by systematically comparing and optimizing techniques for fixation, permeabilization, hybridization, and postprocessing. The new method gives robust, high-resolution labeling in fine anatomical detail, allows co-labeling with fluorescent probes, and is sufficiently sensitive to resolve the expression pattern of a microRNA in planarians. Our WISH methodology not only provides significant advancements over current protocols that make it a valuable asset for the planarian community, but should also find wide applicability in WISH methods used in other systems. PMID:19161223

  20. The Use of Whole-Mount "in Situ" Hybridization to Illustrate Gene Expression Regulation

    ERIC Educational Resources Information Center

    Llamusí, Beatriz; Muñoz-Soriano, Verónica; Paricio, Nuria; Artero, Rubén

    2014-01-01

    "In situ" hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in "Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the…

  1. Fluorescent labels for in situ wet chemistry experiments

    NASA Technical Reports Server (NTRS)

    Kloepfer, J. A.; Nadeau, J. L.

    2003-01-01

    We evaluate a wide selection of dyes and suggest a panel that would be the most likely to succeed in a simple flight instrument with a single excitation laser. We also investigate fluorescent semiconductor quantum dots as additions to or replacements for these organic dyes.

  2. Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes

    NASA Technical Reports Server (NTRS)

    Yu, F. P.; McFeters, G. A.

    1994-01-01

    Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.

  3. Solar cells Improved Hybrid Solar Cells via in situ UV Polymerization

    E-print Network

    Sibener, Steven

    Solar cells Improved Hybrid Solar Cells via in situ UV Polymerization Sanja Tepavcevic, Seth B-enhanced solar energy conversion. By using this simple in situ UV polymerization method that couples mobility of the photoactive layer can be enhanced. 1. Introduction Hybrid solar cells have been developed

  4. [The method of phytoplankton photosynthesis activity in-situ measurement based on light induced fluorescence].

    PubMed

    Liu, Jing; Liu, Wen-qing; Zhao, Nan-jing; Zhang, Yu-jun; Ma, Ming-jun; Yin, Gao-fang; Dai, Pang-da; Wang, Zhi-gang; Wang, Chun-long; Duan, Jing-bo; Yu, Xiao-ya; Fang, Li

    2013-09-01

    According to the phytoplankton fluorescence induction characteristics under different light conditions, chlorophyll fluorescence as a probe for analysis of phytoplankton photosynthesis was studied. The present paper proposed a in-situ measurement method based on the chlorophyll fluorescence values Ft and Fm to get phytoplankton photosynthesis activity, Chlorella vulgaris, microcystis aeruginosa and Cyclotella meneghiniana Kiits were selected as experimental subjects, a comparison test was done between self-developed in-situ measurement system and Water PAM in lab, and the results showed that coefficients between the two methods were 0.9778, 0.8786 and 0.7953. This work provides a rapid and in-situ measurement method for phytoplankton photosynthesis activity. PMID:24369649

  5. Fetal t(5p;21q) misdiagnosed as monosomy 21: A plea for in situ hybridization studies

    SciTech Connect

    Gill, P.; Uhrich, S.; Cheng, E.; Disteche, C.

    1994-10-01

    We report a case of 45,XY,-5,-21,+der (5)t(5;21) (p13 or p14;q11.2 or q21) that was prenatally misdiagnosed as complete monosomy 21 and terminated at 24 weeks of gestation. Subsequent fluorescence in situ hybridization studies with a chromosome 21 painting probe documented the cryptic unbalanced translocation. 17 refs., 2 figs., 1 tab.

  6. Digoxigenin-labeled in situ hybridization for the detection of Streptococcus suis DNA in polyserositis and a comparison with biotinylated in situ hybridization.

    PubMed

    Kang, Ikjae; Seo, Hwi Won; Park, Changhoon; Oh, Yeonsu; Lee, Jeehoon; You, Ok Heui; Kim, Sung-Hoon; Gottschalk, Marcelo; Chae, Chanhee

    2014-01-01

    The objective of this study was to develop digoxigenin-labeled in situ hybridization (ISH) for the detection of Streptococcus suis in naturally infected pigs with polyserositis and to compare it with biotinylated ISH. Digoxigenin-labeled hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis and microcolonies in the blood vessels. Mock hybridization showed no hybridization signals for endogenous digoxigenin. Biotinylated hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis. However, similar hybridization signals were also observed in the fibrous inflammatory area using mock hybridization for endogenous biotin. The present study demonstrated that digoxigenin-labeled ISH is a valuable diagnostic tool for specific detection of S. suis in polyserositic tissues without nonspecific reactions compared with biotinylated ISH. PMID:23985415

  7. Analysis of chromosome segregation during mammalian meiosis using combined immunofluorescence and fluorescence in situ hubridization

    SciTech Connect

    Hunt, P.A.; Embury, P.B.; Mroz, K.M.

    1994-09-01

    Meiotic non-disjunction is thought to occur in 10-20% of all human oocytes, making this the most common genetic abnormality in our species. Aberrant recombination has been implicated in the genesis of these errors; however, direct studies of the meiotic process have been hampered by the lack of material and appropriate technology. We have developed a technique for the evaluation of meiosis in intact mammalian oocytes that combines immunofluorescence and fluorescence in situ hybridization (FISH). This allows for simultaneous, 3-dimensional visualization of the meiotic spindle, the alignment of the chromosomes on the spindle, and the placement of specific chromosomes. We have used this technology to follow meiotic progression in oocytes from XO female mice to evaluate the behavior of an unsynapsed chromosome during mammalian meiosis. Perturbations in chromosome behavior are evident early in meiosis: during the formation of the first meiotic spindle, the univalent X chromosome is properly positioned. With the onset of anaphase, the single X chromosome most commonly segregates as an intact chromosome, although equational segregation of the X chromatids is seen in a significant minority (approximately 20%) of oocytes. These observations demonstrate that failure of pairing/recombination can result in segregation of sister chromatids at meiosis I. This has obvious implications for human non-disjunction, much of which is thought to be due to recombination deficiencies; accordingly, we are now extending our studies to include analyses of human oocytes.

  8. Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy

    PubMed Central

    Prabhakaran, Venkateshkumar; Arges, Christopher G.; Ramani, Vijay

    2012-01-01

    A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate. PMID:22219367

  9. Genomic Origin and Organization of the Allopolyploid Primula egaliksensis Investigated by in situ Hybridization

    PubMed Central

    Guggisberg, Alessia; Baroux, Célia; Grossniklaus, Ueli; Conti, Elena

    2008-01-01

    Background and Aims Earlier studies have suggested that the tetraploid Primula egaliksensis (2n = 40) originated from hybridization between the diploids P. mistassinica (2n = 18) and P. nutans (2n = 22), which were hypothesized to be the maternal and paternal parent, respectively. The present paper is aimed at verifying the hybrid nature of P. egaliksensis using cytogenetic tools, and to investigate the extent to which the parental genomes have undergone genomic reorganization. Methods Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with ribosomal DNA (rDNA) probes, together with sequencing of the internal transcribed spacer (ITS) region of the rDNA, were used to identify the origin of P. egaliksensis and to explore its genomic organization, particularly at rDNA loci. Key Results GISH showed that P. egaliksensis inherited all chromosomes from P. mistassinica and P. nutans and did not reveal major intergenomic rearrangements between the parental genomes (e.g. interchromosomal translocations). However, karyological comparisons and FISH experiments suggested small-scale rearrangements, particularly at rDNA sites. Primula egaliksensis lacked the ITS-bearing heterochromatic knobs characteristic of the maternal parent P. mistassinica and maintained only the rDNA loci of P. nutans. These results corroborated sequence data indicating that most ITS sequences of P. egaliksensis were of the paternal repeat type. Conclusions The lack of major rearrangements may be a consequence of the considerable genetic divergence between the putative parents, while the rapid elimination of the ITS repeats from the maternal progenitor may be explained by the subterminal location of ITS loci or a potential role of nucleolar dominance in chromosome stabilization. These small-scale rearrangements may be indicative of genome diploidization, but further investigations are needed to confirm this assumption. PMID:18308718

  10. Understanding aquatic microbial processes using EEM's and in-situ fluorescence sensors

    NASA Astrophysics Data System (ADS)

    Fox, Bethany; Attridge, John; Rushworth, Cathy; Cox, Tim; Anesio, Alexandre; Reynolds, Darren

    2015-04-01

    The diverse origin of dissolved organic matter (DOM) in aquatic systems is well documented within the literature. Previous literature indicates that coloured dissolved organic matter (CDOM) is, in part, transformed by aquatic microbial processes, and that dissolved organic material derived from a microbial origin exhibits tryptophan-like fluorescence. However, this phenomenon is not fully understood and very little data is available within the current literature. The overall aim of our work is to reveal the microbial-CDOM interactions that give rise to the observed tryptophan-like fluorescence. The work reported here investigates the microbial processes that occur within freshwater aquatic samples, as defined by the biochemical oxygen demand (BOD) test, as a function of the T1 peak (?ex/em 280/330-370 nm). A series of standard water samples were prepared using glucose, glutamic acid, BOD dilution water and a bacterial seed (Cole-Parmer BOD microbe capsules). Samples were spiked with CDOM (derived from an environmental water body) and subjected to time resolved BOD analysis and as excitation-emission fluorescence spectroscopy. All EEM spectral data was interrogated using parallel factor analysis (PARAFAC) in an attempt to determine the presence and dominance (relative intensities) of the CDOM-related and T1-related fluorophores within the samples. In-situ fluorescence sensors (Chelsea Technologies Group Ltd.) were also used to monitor the T1 fluorescence peak (UviLux Tryptophan) and the CDOM fluorescence peak (UviLux CDOM) during experiments. Tryptophan-like fluorescence was observed (albeit transient) in both spiked and un-spiked standard water samples. By furthering our understanding of aquatic organic matter fluorescence, its origin, transformation, fate and interaction with aquatic microbiological processes, we aim to inform the design of a new generation in-situ fluorescence sensor for the monitoring of aquatic ecosystem health.

  11. Quantifying the photothermal efficiency of gold nanoparticles using tryptophan as an in situ fluorescent thermometer.

    PubMed

    Chiu, Ming-Jui; Chu, Li-Kang

    2015-07-14

    The photothermal efficiencies, denoting the efficiency of transducing incident light to heat, of gold nanoparticles of different diameters (? = 22-86 nm) were quantified upon exposure at 532 nm. The fluorescence of tryptophan at 300-450 nm upon 280 nm excitation serves as an in situ fluorescent thermometer to illustrate the evolution of the average temperature change in the heating volume of the nanoparticle solution. The fluorescence intensity decreases as the temperature increases, having a linear gradient of 2.05% fluorescence decrease per degree Celsius increment from 20 to 45 °C. The presence of gold nanoparticles at the nM level does not perturb the temperature-dependent fluorescence of tryptophan in terms of fluorescence contour and temperature response. The heating volume was defined by overlapping the collimated 532 nm laser (? = 0.83 mm) for exciting the nanoparticles and the 280 nm continuous-wave beam (? = 0.81 mm) for exciting tryptophan in a 2 mm × 2 mm square tube, and the fluorescence was collected perpendicularly to the collinear alignment. This method has satisfactory reproducibility and a sufficient temperature detectivity of 0.2 °C. The profiles of the average temperature evolution of the mixtures containing nanoparticles and tryptophan were derived from the evolution of fluorescence and analyzed using collective energy balancing. The relative photothermal efficiencies for different sizes of gold nanoparticles with respect to the 22 nm nanoparticle agree with those predicted using Mie theory. The employment of tryptophan as a fluorescent thermometer not only provides an in situ tool to monitor the photothermal effect of nanostructures but is also applicable to thermal imaging in biological applications. PMID:26068797

  12. Regulatory pathway analysis by high-throughput in situ hybridization

    SciTech Connect

    Visel, Axel; Carson, James P.; Oldekamp, Judit; Warnecke, Marei; Jakubcakova, Vladimira; Zhou, Xunlei; Shaw, Chad; Alvarez-Bolado, Gonzalo; Eichele, Gregor

    2007-10-01

    Automated in situ hybridization (ISH) permits construction of comprehensive atlases of gene expression patterns in mammals. When web-accessible, such atlases become searchable digital expression maps of individual genes and offer an entryway to elucidate genetic interactions and signaling pathways. An atlas housing ~1,000 spatial gene expression patterns of the mid-gestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising 90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This ordered hundreds of complex expression patterns into a matrix that reflected the embryonic architecture and the relatedness of patterns of expression. Clustering yielded twelve distinct groups of expression pattern. Because of similarity of expression patterns within a group, members of this group may be components of regulatory cascades. We focused on one group, which is composed of 83 genes, including Pax6, an evolutionary conserved transcriptional master mediator of the development. Using functional studies, ISH on Pax6-deficient embryos and Pax6 binding site identification and validation by means of electromobility shift assays, we identified numerous genes that are transcriptionally regulated by Pax6. Hence cluster analysis of annotated gene expression patterns obtained by robotic ISH is an entryway for identification of components of signaling cascades in mammals.

  13. Portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection

    NASA Astrophysics Data System (ADS)

    Mota, Alessandro D.; Rossi, Giuliano; de Castro, Guilherme Cunha; Ortega, Tiago A.; de Castro N., Jarbas C.

    2014-02-01

    In this work, the development of a portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection is presented. The equipment consists of an excitation blue LED light source, a commercial miniature spectrometer and embedded software. Measurements of healthy, HLB-symptomatic and HLB-asymptomatic citrus leafs were performed. Leafs were excited with the blue LED and their fluorescence spectra collected. Embedded electronics and software were responsible for the spectrum processing and classification via partial least squares regression. Global success rates above 80% and 100% distinction of healthy and HLB-symptomatic leafs were obtained.

  14. In Situ Biosynthesis of Fluorescent Platinum Nanoclusters: Toward Self-Bioimaging-Guided Cancer Theranostics.

    PubMed

    Chen, Donghua; Zhao, Chunqiu; Ye, Jing; Li, Qiwei; Liu, Xiaoli; Su, Meina; Jiang, Hui; Amatore, Christian; Selke, Matthias; Wang, Xuemei

    2015-08-19

    Among the noble-metal clusters, very few reports about platinum clusters were used as bioimaging probes of tumors except as a reducing catalyst. It is first established herein that the biocompatible platinum nanoclusters are spontaneously biosynthesized by cancerous cells (i.e., HepG2 (human hepatocarcinoma), A549 (lung cancer), and others) rather than noncancerous cells (i.e., L02 (human embryo liver cells)) when incubated with micromolar chloroplatinic acid solutions. These in situ biosynthesized platinum nanoclusters could be readily realized in a biological environment and emit a bright fluorescence at 460 nm, which could be further utilized to facilitate an excellent cancer-cell-killing efficiency when combined with porphyrin derivatives for photothermal treatment. This raises the possibility of providing a promising and precise bioimaging strategy for specific fluorescent self-biomarking of tumor locations and realizing fluorescence imaging-guided photothermal therapy of tumors. PMID:26227621

  15. Amplification of chromosomal DNA in situ

    DOEpatents

    Christian, Allen T. (Tracy, CA); Coleman, Matthew A. (Livermore, CA); Tucker, James D. (Livermore, CA)

    2002-01-01

    Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

  16. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RIBOSOMAL RNA-TARGETED OLIGONUCLEOTIDE PROBE

    EPA Science Inventory

    A fluroescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligonu...

  17. In situ microbial detection in Mojave Desert soil using native fluorescence.

    PubMed

    Smith, H D; Duncan, A G; Neary, P L; Lloyd, C R; Anderson, A J; Sims, R C; McKay, C P

    2012-03-01

    We report on the use of a portable instrument for microbial detection in the Mojave Desert soil and the potential for its use on Mars. The instrument is based on native fluorescence and employs four excitation wavelengths combined with four emission wavelengths. A soil dilution series in which known numbers of Bacillus subtilis spores were added to soil was used to determine the sensitivity of the instrument. We found that the fluorescence of the biological and organic components of the desert soil samples studied can be as strong as the fluorescence of the mineral component of these soils. Using the calibration derived from B. subtilis spores, we estimated that microbial content at our primary sampling site was 10(7) bacteria per gram of soil, a level confirmed by phospholipid fatty acid analysis. At a nearby site, but in a slightly different geological setting, we tested the instrument's ability to map out microbial concentrations in situ. Over a ?50 m diameter circle, soil microbial concentrations determined with the B. subtilis calibration indicate that the concentrations of microorganisms detected varies from 10(4) to 10(7) cells per gram of soil. We conclude that fluorescence is a promising method for detecting soil microbes in noncontact applications in extreme environments on Earth and may have applications on future missions to Mars. PMID:22352702

  18. miRNA in situ hybridization in formaldehyde and EDC-fixed tissues.

    PubMed

    Pena, John T G; Sohn-Lee, Cherin; Rouhanifard, Sara H; Ludwig, Janos; Hafner, Markus; Mihailovic, Aleksandra; Lim, Cindy; Holoch, Daniel; Berninger, Philipp; Zavolan, Mihaela; Tuschl, Thomas

    2009-02-01

    MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5' phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH. PMID:19137005

  19. Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics.

    PubMed

    Franco, Juliana C; Gonçalves, Grasiele; Souza, Monique S; Rosa, Samantha B C; Thiegue, Larissa M; Atvars, Teresa D Z; Rosa, Paulo T V; Nome, René A

    2013-12-16

    We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy. PMID:24514660

  20. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.

    PubMed

    Lee, Je Hyuk; Daugharthy, Evan R; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C; Terry, Richard; Turczyk, Brian M; Yang, Joyce L; Lee, Ho Suk; Aach, John; Zhang, Kun; Church, George M

    2015-03-01

    RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d. PMID:25675209

  1. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

    PubMed Central

    Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Terry, Richard; Turczyk, Brian M.; Yang, Joyce L.; Lee, Ho Suk; Aach, John; Zhang, Kun; Church, George M.

    2014-01-01

    RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d. PMID:25675209

  2. Rate of in situ Shattercane x Sorghum Hybridization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum (Sorghum bicolor subsp. bicolor) can interbreed with its close weedy relative shattercane (S. bicolor subsp. drummondii). The introduction of traits from sorghum into a shattercane population could contribute to the invasiveness of the wild shattercane population. An in situ experiment was...

  3. miRNA in situ hybridization in circulating tumor cells - MishCTC

    PubMed Central

    Ortega, Francisco G.; Lorente, Jose A.; Garcia Puche, Jose L.; Ruiz, Maria P.; Sanchez-Martin, Rosario M.; de Miguel-Pérez, Diego; Diaz-Mochon, Juan J.; Serrano, Maria J.

    2015-01-01

    Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype. PMID:25777797

  4. Identification of a tumor marker chromosome by flow sorting, DNA amplification in vitro, and in situ hybridization of the amplified product

    SciTech Connect

    Boschman, G.; Rens, W.; Slater, R.; Aten, J. ); Buys, C.; Veen, A. van der; Osinga, J. )

    1993-01-01

    A method combining flow sorting and molecular cytogenetic techniques was used to identify an unknown marker chromosome in the bladder tumor cell line J82. The marker chromosome was isolated by dual parameter sorting after staining with Hoechst 33258 and chromomycin [Lambda]3. DNA amplification of 300 isolated chromosomes by polymerase chain reaction using the Alu-primer Bk33 and the Lines-primer LH5 was carried out. The DNA was labelled using Bio-11-dUTP and applied to human lymphocyte metaphase cells in a suppressive in situ hybridization procedure. Fluorescence was visible over chromosome 20 and over the distal one-half of 6p. Together the fluorescent regions accounted for only 60% of the marker length, indicating a possible duplication of chromosome 20 material. This was subsequently confirmed by applying bicolor in-situ hybridization using chromosome 6- and 20-specific DNA libraries to metaphase cells of the J82 culture.

  5. Synergetic spin crossover and fluorescence in one-dimensional hybrid complexes.

    PubMed

    Wang, Chun-Feng; Li, Ren-Fu; Chen, Xue-Yuan; Wei, Rong-Jia; Zheng, Lan-Sun; Tao, Jun

    2015-01-26

    Hybrid materials integrated with a variety of physical properties, such as spin crossover (SCO) and fluorescence, may show synergetic effects that find applications in many fields. Herein we demonstrate a promising post-synthetic approach to achieve such materials by grafting fluorophores (1-pyrenecarboxaldehyde and Rhodamine?B) on one-dimensional SCO Fe(II) structures. The resulting hybrid materials display expected one-step SCO behavior and fluorescent properties, in particular showing a coupling between the transition temperature of SCO and the temperature where the fluorescent intensity reverses. Consequently, synergetic effect between SCO and fluorescence is incorporated into materials despite different fluorophores. This study provides an effective strategy for the design and development of novel magnetic and optical materials. PMID:25504738

  6. A PDMS-Based Cylindrical Hybrid Lens for Enhanced Fluorescence Detection in Microfluidic Systems

    PubMed Central

    Lin, Bor-Shyh; Yang, Yu-Ching; Ho, Chong-Yi; Yang, Han-Yu; Wang, Hsiang-Yu

    2014-01-01

    Microfluidic systems based on fluorescence detection have been developed and applied for many biological and chemical applications. Because of the tiny amount of sample in the system; the induced fluorescence can be weak. Therefore, most microfluidic systems deploy multiple optical components or sophisticated equipment to enhance the efficiency of fluorescence detection. However, these strategies encounter common issues of complex manufacturing processes and high costs. In this study; a miniature, cylindrical and hybrid lens made of polydimethylsiloxane (PDMS) to improve the fluorescence detection in microfluidic systems is proposed. The hybrid lens integrates a laser focusing lens and a fluorescence collecting lens to achieve dual functions and simplify optical setup. Moreover, PDMS has advantages of low-cost and straightforward fabrication compared with conventional optical components. The performance of the proposed lens is first examined with two fluorescent dyes and the results show that the lens provides satisfactory enhancement for fluorescence detection of Rhodamine 6G and Nile Red. The overall increments in collected fluorescence signal and detection sensitivity are more than 220% of those without lens, and the detection limits of Rhodamine 6G and Nile red are lowered to 0.01 ?g/mL and 0.05 ?g/mL, respectively. The hybrid lens is further applied to the detection of Nile red-labeled Chlorella vulgaris cells and it increases both signal intensity and detection sensitivity by more than 520%. The proposed hybrid lens also dramatically reduces the variation in detected signal caused by the deviation in incident angle of excitation light. PMID:24531300

  7. Progress in molecular diagnosis of Charcot-Marie-Tooth-disease type 1 (CMT 1, HMSN I) and hereditary neuropathy with liability to pressure palsies (HNPP) by fluorescence in situ hybridization (FISH)-detection of a potential genetic mosaicism

    SciTech Connect

    Bathke, K.; Liehr. T.; Ekici, A.

    1994-09-01

    We tested 20 CMT 1 patients characterized according to the criteria of the European CMT consortium by Southern hybridization of MspI restricted genomic DNA with probes pVAW409R1, pVAW412Hec and pEW401HE. In 11 of the 20 CMT 1 cases (55%), we observed a duplication in 17q11.2; one patient had a dinucleotide insertion in exon 6 of the PO-gene (5%). One HNPP case had a typical 17p11.2 deletion. Analysis of CA-repeats was performed with primers RM11GT and Mfd41; SSCP-analysis of the PO, PMP22 and Cx32-genes is in progress. FISH was carried out with probe pVAW409R1. 125 interphase nuclei were analyzed for each proband by counting the signals per nucleus. Normal cells show a characteristic distribution of signals: 1 signal in 5.9% of nuclei, 2 in 86.3% and 3 in 7.8%. A duplication is indicated by a shift to 3 signals in more than approximately 60% and 2 in less than 25% of the nuclei. In contrast, the 17p11.2 deletion of the HNPP patient shifts to 82.4% of nuclei with a single hybridization signal versus 14.4% with 2 signals. We detected one case with significantly abnormal distribution of interphase nuclei hybridization signals compared to cultures of normal cells and to those with 17p11.2 duplication or deletion: 3.2% nuclei revealed 1 signal, 48.0% two signals and 48.8% 3 signals, indicating a pathogenic but moderate dosis increase compared to the throughout duplicated cases. FISH with probe pVAW409R1 is a versatile tool to detect the HNPP deletion both in interphase nuclei and in metaphase chromosomes. In CMT 1 disease interphase nuclei are required for FISH analysis due to the small duplication of 1.5 Mbp. In contrast to Southern techniques, FISH is able to detect genetic mosaicism.

  8. Fluorescence spectroscopy of collagen crosslinking: non-invasive and in situ evaluation of corneal stiffness

    NASA Astrophysics Data System (ADS)

    Franco, Walfre; Ortega-Martinez, Antonio; Zhu, Hong; Wang, Ruisheng; Kochevar, Irene E.

    2015-03-01

    Collagen is a long fibrous structural protein that imparts mechanical support, strength and elasticity to many tissues. The state of the tissue mechanical environment is related to tissue physiology, disease and function. In the cornea, the collagen network is responsible for its shape and clarity; disruption of this network results in degradation of visual acuity, for example in the keratoconus eye disease. The objective of the present study is to investigate the feasibility of using the endogenous fluorescence of collagen crosslinks to evaluate variations in the mechanical state of tissue, in particular, the stiffness of cornea in response to different degrees of photo-crosslinking or RGX treatment—a novel keratoconus treatment. After removing the epithelium, rabbit corneas were stained with Rose Bengal and then irradiated with a 532 nm solid-state laser. Analysis of the excitation spectra obtained by fluorescence spectroscopy shows a correlation between the fluorescence intensity at 370/460 nm excitation/emission wavelengths and the mechanical properties. In principle, it may be feasible to use the endogenous fluorescence of collagen crosslinks to evaluate the mechanical stiffness of cornea non-invasively and in situ.

  9. Genetically assembled fluorescent biosensor for in situ detection of bio-synthesized alkanes.

    PubMed

    Wu, Wei; Zhang, Lei; Yao, Lun; Tan, Xiaoming; Liu, Xufeng; Lu, Xuefeng

    2015-01-01

    Construction of highly efficient microbial cell factories producing drop-in biofuel alkanes is severely limited due to the lack of a fast detection method against alkanes. Here we first developed a sensitive fluorescent biosensor for rapid and in situ monitoring of intracellular alkane synthesis. Using GFP as reporter, the biosensor could actively respond to the intracellular alkane products, especially for the mid- and long-chain alkanes synthesized in the recombinant Escherichia coli and give a concentration-dependent fluorescence response. Our results also suggested the feasibility of developing high-throughput strategies basing on the alkane biosensor device in E. coli, and thus will greatly facilitate the application of directed evolution strategies to further improve the alkane-producing microbial cell factories. PMID:26039923

  10. In-situ detection of viral nucleic acids using fluorescent probes

    NASA Astrophysics Data System (ADS)

    Donovan, Richard M.

    1990-07-01

    The objective of this work was to develop and improve technologies in cytometry and molecular biology for the specific in situ detection of viral nucleic acids. The major application for this system was the detection and measurement of individual cells stained specifically for the Human Immunodeficiency Virus (HIV) in patients with Acquired Immune Deficiency Syndrome (AIDS). Staining procedures used nucleic acid either directly or indirect labeled with enzymes or fluorescent probes. A cytometry system was used to acquire digitized images of labeled cells and determine their individual staining density or intensity. Efforts are underway to improve the sensitivity of these assays using time-resolved methods.

  11. In-Situ Silver Acetylide Silver Nitrate Explosive Deposition Measurements Using X-Ray Fluorescence.

    SciTech Connect

    Covert, Timothy T.

    2014-09-01

    The Light Initiated High Explosive facility utilized a spray deposited coating of silver acetylide - silver nitrate explosive to impart a mechanical shock into targets of interest. A diagnostic was required to measure the explosive deposition in - situ. An X - ray fluorescence spectrometer was deployed at the facility. A measurement methodology was developed to measure the explosive quantity with sufficient accuracy. Through the use of a tin reference material under the silver based explosive, a field calibration relationship has been developed with a standard deviation of 3.2 % . The effect of the inserted tin material into the experiment configuration has been explored.

  12. Coexistence of t(15;17) and t(15;16;17) detected by fluorescence in situ hybridization in a patient with acute promyelocytic leukemia: A case report and literature review

    PubMed Central

    ZHANG, RUI; KIM, YOUNG-MI; WANG, XIANFU; LI, YAN; PANG, HUI; LEE, JI-YUN; LI, SHIBO

    2014-01-01

    Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), which results in the fusion of the promyelocytic leukemia (PML) gene at 15q22 with the retinoic acid ?-receptor (RARA) gene at 17q21. The current study presents the case of a 54-year-old female with APL carrying the atypical PML/RARA fusion signal due to a novel complex variant translocation t(15;16;17)(q22;q24;q21), as well as the classical PML/RARA fusion signal. Subsequent array comparative genomic hybridization revealed somatic, cryptic deletions on 3p25.3, 8q23.1 and 12p13.2-p13.1, and a duplication on 8q11.2; however, no genetic material loss or gain was observed in the breakpoint regions of chromosomes 15, 16 or 17. To the best of our knowledge, this is the first report of the coexistence of two abnormal clones, one classical and one variant, presenting simultaneously in addition to cryptic chromosome segmental imbalances in an adult APL patient. PMID:25120648

  13. Whole Mount Drosophila Embryo In Situ Hybridization with RNA probes Leslie Vosshall

    E-print Network

    1 Whole Mount Drosophila Embryo In Situ Hybridization with RNA probes 2/5/2001 Leslie Vosshall DAY to the top with wash solution and place embryos on a nutator during the washing period. 1. Prepare fixed embryos according to attached protocol until 70% ethanol step. 2. Remove an aliquot of embryos sufficient

  14. ELF{trademark}: A new fluorogenic alkaline phosphatase substrate for nonisotopic mRNA in situ hybridization

    SciTech Connect

    Singer, V.; Paragas, V.; Zhang, Y.Z.

    1994-09-01

    Molecular Probes` researchers have developed a novel alkaline phosphatase substrate that gives rise to a bright yellow-green fluorescent precipitate at the site of enzymatic activity. We refer to this process as ELF, for Enzyme-Labeled Fluorescence. We found that the ELF substrate is an ideal tool for mRNA in situ hybridization. Not only are ELF signals much brighter than those generated using conventional fluorophores, resulting in film exposure times approximately one-fortieth as long as those needed for fluorescein-labeled probes or secondary detection reagents, but the ELF signal is also many-fold more photostable. The ELF signal develops in seconds to minutes, in contrast to radioactive detection, which requires days to weeks for adequate signal development. In addition, because the ELF precipitate has a Stokes shift of greater than 100 nm, we found that ELF signals can be easily distinguished from sample autofluorescence and from signals arising from other fluorescent probes or counterstrains. Also, unlike signals from chromogenic enzyme substrates, the fluorescent ELF signals are easily distinguished from pigmented tissues, even in zebrafish whole-mount embryos.

  15. Demonstration of tissue-specific gene expression by in situ hybridization

    SciTech Connect

    Angerer, L.M.; Cox, K.H.; Angerer, R.C.

    1987-01-01

    In situ hybridization has emerged as a valuable tool for the identification of individual cells expressing specific genes. Recently, methods have become sufficiently sensitive to detect mRNAs present at the level of only a few molecules per cell. When mRNAs are expressed in only a small fraction of the cells in a mixed population, in situ hybridization may be the most sensitive nucleic acid hybridization technique available. The authors' laboratory has shown that antisense RNA probes offer a unique combination of advantages for detection of individual mRNAs by in situ hybridization. Most importantly, antisense probes provide a large increase in sensitivity due to the absence of competing probe self-reassociation. The high stability of RNA-RNA duplexes allows use of higher post hybridization wash temperatures to achieve a given fidelity of base pairing (stringency), which also reduced backgrounds. RNA transcripts can be synthesized from truncated templates such that they are essentially devoid of vector sequences. This sequence purity maximizes the signal to noise ratio since lower probe concentrations are required to saturate target RNAs.

  16. Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG-Labeled

    E-print Network

    Ruby, Edward G.

    Protocol Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG- Labeled Riboprobes: I. DNA

  17. Development of a fluorescent in situ method for visualization of enteric viruses.

    PubMed

    Rawsthorne, Helen; Phister, Trevor G; Jaykus, Lee-Ann

    2009-12-01

    Studying the interactions between enteric pathogens and their environment is important to improving our understanding of their persistence and transmission. However, this remains challenging in large part because of difficulties associated with tracking pathogens in their natural environment(s). In this study, we report a fluorescent labeling strategy which was applied to murine norovirus (MNV-1), a human norovirus surrogate, and hepatitis A virus (HAV). Specifically, streptavidin-labeled Quantum dots (Q-Dots) were bound to biotinylated capsids of MNV-1 and HAV (bio-MNV-1 and bio-HAV); the process was confirmed by using a sandwich-type approach in which streptavidin-bound plates were reacted with biotinylated virus followed by a secondary binding to Q-Dots with an emission range of 635 to 675 nm (Q-Dots 655). The assay demonstrated a relative fluorescence of 528 +/- 48.1 and 112 +/- 8.6 for bio-MNV-1 and control MNV-1, respectively. The biotinylation process did not impact virus infectivity, nor did it interfere with the interactions between the virus and host cells or model produce items. Using fluorescent microscopy, it was possible to visualize both bio-HAV and bio-MNV-1 attached to the surfaces of permissive mammalian cells and green onion tissue. The method provides a powerful tool for the labeling and detection of enteric viruses (and their surrogates) which can be used to track virus behavior in situ. PMID:19854924

  18. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.

    PubMed Central

    Pinkel, D; Straume, T; Gray, J W

    1986-01-01

    This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe. Interspecies translocations were detected easily at metaphase. The human-specific fluorescence intensity from cell nuclei and chromosomes was proportional to the amount of target human DNA. Human Y chromosomes were fluorescently stained in metaphase and interphase nuclei by using a 0.8-kilobase DNA probe specific for the Y chromosome. Cells from males were 40 times brighter than those from females. Both Y chromosomal domains were visible in most interphase nuclei of XYY amniocytes. Human 28S ribosomal RNA genes on metaphase chromosomes were distinctly stained by using a 1.5-kilobase DNA probe. Images PMID:3458254

  19. In situ measurements of subsurface contaminants with a multi-channel laser-induced fluorescence system

    NASA Astrophysics Data System (ADS)

    Wu Pepper, Jane; Wright, Andrew O.; Kenny, Jonathan E.

    2002-01-01

    A new multi-channel laser-induced fluorescence (LIF) probe with novel optical fiber probe geometry has been designed and integrated into a cone penetrometer testing (CPT) system for in situ contamination detection. The system is capable of collecting excitation and emission matrices (EEMs) of subsurface contaminants as a function of depth in seconds. Compared to our previous multi-channel LIF-CPT system, the new system is faster and more compact, with reduced probe size and sampling area. This article describes the first field demonstration of the system at Hanscom Air Force Base, Massachusetts. One contaminated site within the base was characterized through in situ measurements of 26 LIF-CPT pushes. To validate the LIF results, core samples taken at five locations were analyzed by both on-site LIF measurements and by off-site laboratory analyses with EPA methods. The comparison of the LIF and laboratory results is presented, along with the results of the in situ measurements.

  20. In situ measurements of subsurface contaminants with a multi-channel laser-induced fluorescence system.

    PubMed

    Pepper, Jane Wu; Wright, Andrew O; Kenny, Jonathan E

    2002-01-15

    A new multi-channel laser-induced fluorescence (LIF) probe with novel optical fiber probe geometry has been designed and integrated into a cone penetrometer testing (CPT) system for in situ contamination detection. The system is capable of collecting excitation and emission matrices (EEMs) of subsurface contaminants as a function of depth in seconds. Compared to our previous multi-channel LIF-CPT system, the new system is faster and more compact, with reduced probe size and sampling area. This article describes the first field demonstration of the system at Hanscom Air Force Base, Massachusetts. One contaminated site within the base was characterized through in situ measurements of 26 LIF-CPT pushes. To validate the LIF results, core samples taken at five locations were analyzed by both on-site LIF measurements and by off-site laboratory analyses with EPA methods. The comparison of the LIF and laboratory results is presented, along with the results of the in situ measurements. PMID:11808737

  1. West Nile Virus Infection in Horses: Detection by Immunohistochemistry, In Situ Hybridization, and ELISA.

    PubMed

    Toplu, N; O?uzo?lu, T Ç; Ural, K; Albayrak, H; Ozan, E; Ertürk, A; Epikmen, E T

    2015-11-01

    This study describes the clinicopathologic findings in naturally occurring West Nile virus (WNV) infection in horses. WNV was diagnosed in a foal by immunohistochemical and in situ hybridization methods, and the presence of WNV antibodies was detected in 5 other horses with clinical signs suggestive of WNV infection. At necropsy of the foal, lymph nodes were edematous and enlarged, and the intestines showed diffuse congestion and focal hemorrhages. The most significant histologic lesions in this case were nonsuppurative meningoencephalomyelitis, particularly in the brainstem and spinal cord. Identification of viral RNA by in situ hybridization and viral antigen by immunohistochemistry was concentrated primarily in nerve fibers, glial cells, and their processes in brainstem and spinal cord and, to a lesser extent, within the cerebral hemispheres and cerebellum. PMID:25677341

  2. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    PubMed Central

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno; Madureira, Pedro; Ferreira, Rui Manuel; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2015-01-01

    In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization. PMID:25915865

  3. Assessing HER2 amplification in breast cancer: findings from the Australian In Situ Hybridization Program.

    PubMed

    Bilous, Michael; Morey, Adrienne L; Armes, Jane E; Bell, Richard; Button, Peter H; Cummings, Margaret C; Fox, Stephen B; Francis, Glenn D; Waite, Brigid; McCue, Glenda; Raymond, Wendy A; Robbins, Peter D; Farshid, Gelareh

    2012-07-01

    In August 2006, the Australian government approved subsidized trastuzumab therapy for human epidermal growth factor receptor 2 (HER2)-positive early breast cancer, and it was mandated that HER2 testing should be performed using in situ hybridization (ISH) rather than immunohistochemistry (IHC). Here we review results of the first regulated, nationwide program to provide HER2 ISH testing for all newly diagnosed breast cancer patients, with a particular emphasis on cases where IHC and ISH results were discordant. Data from all laboratories participating in the program were collated. Cases with an equivocal ISH test result [by chromogenic ISH (CISH) or silver ISH (SISH)] were tested centrally by fluorescence ISH. Most laboratories also performed HER2 IHC, and 200 cases with discordant IHC and ISH results were selected for further analysis in a central laboratory. A total of 26 laboratories were involved and 53,402 tests were reported. Over a 4-year period the HER2 positivity rate decreased for primary cancers from 23.8 to 14.6 %, but remained relatively constant for samples from metastases. Average ISH reporting times were <5 days for all yearly reporting periods. Test-repeat rates decreased for CISH (8.9-3.6 %) and SISH (13.7-8.4 %). Only 12 of 196 cases remained discordant after retesting in a central laboratory. These findings demonstrate the successful implementation of a regulated, national program that continues to collect data on HER2 status. The results also highlight the differences in IHC interpretation between local laboratories and a central, more experienced, laboratory. This model could be used to establish future biomarker-testing programs in other countries. PMID:22678156

  4. miRNA in situ hybridization in mammalian tissues fixed with formaldehyde and EDC

    PubMed Central

    Pena, John T. G.; Sohn-Lee, Cherin; Rouhanifard, Sara H.; Ludwig, Janos; Hafner, Markus; Mihailovic, Aleksandra; Lim, Cindy; Holoch, Daniel; Berninger, Philipp; Zavolan, Mihaela; Tuschl, Thomas

    2010-01-01

    MicroRNAs (miRNAs) are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in significant miRNA loss from mouse tissue sections, which can be prevented by fixation with 1–ethyl–3–(3–dimethylaminopropyl) carbodiimide (EDC) that irreversibly immobilizes the miRNA at its 5' phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid (LNA) probes by recording nucleic acid melting temperature during ISH. PMID:19137005

  5. Mapping of the gene encoding the melanocortin-1 ([alpha]-melanocyte stimulating hormone) receptor (MC1R) to human chromosome 16q24. 3 by fluorescence in situ hybridization

    SciTech Connect

    Gantz, I.; Yamada, Tadataka; Tashiro, Takao; Konda, Yoshitaka; Shimoto, Yoshimasa; Miwa, Hiroto; Trent, J.M. )

    1994-01-15

    [alpha]-Melanocyte stimulating hormone ([alpha]-MSH), a hormone originally named for its ability to regulate pigmentation of melanocytes, is a 13-amino-acid post-translational product of the pro-opiomelanocortin (POMC) gene. [alpha]-MSH and the other products of POMC processing, which share the core heptapeptide amino acid sequence Met-Glu (Gly)-His-Phe-Arg-Trp-Gly (Asp), the adrenocorticotropic hormone (ACTH), [beta]-MSH, and [gamma]-MSH, are collectively referred to as melanocortins. While best known for their effects on the melanocyte (pigmentation) and adrenal cortical cells (steroidogenesis), melanocortins have been postulated to function in diverse activities, including enhancement of learning and memory, control of the cardiovascular system, analgesia, thermoregulation, immunomodulation, parturition, and neurotrophism. To identify the chromosomal band encoding the human melanocortin-1 receptor gene, 1 [mu]g of an EMBL clone coding region of the human MC1R and approximately 15 kb of surrounding DNA was labeled with biotin and hybridized to human metaphase chromosomes as previously described. The results indicate that the human MC1R gene is localized to 16q24.3. 15 refs., 1 fig.

  6. Continuous in situ fluorescence imaging of an ultracold Fermi gas in an optical lattice

    NASA Astrophysics Data System (ADS)

    Anderson, Rhys; Edge, Graham; Day, Ryan; Nino, Daniel; Trotzky, Stefan; Thywissen, Joseph

    2015-05-01

    We demonstrate continuous in situ fluorescence imaging of ultracold fermionic 40K atoms held in a three-dimensional optical lattice with 527 nm periodicity. Using a 4S-4P1/2 grey molasses cooling scheme with a coherent dark state, we obtain a photon scattering rate exceeding 1 kHz while measuring a steady-state population of the vibrational ground state of 80%. Collecting the scattered photons through a 200 ?m thin sapphire vacuum window and into a microscope objective allows us to image the in situ density distribution of the lattice gas. Spatially selective state manipulation is used to reduce the number of occupied lattice planes along the imaging direction, as well as to create density patterns along the transverse direction. We characterize the performance of the imaging protocol over a wide range of parameters. For larger-than-unity site occupation we observe efficient removal of atoms due to light-assisted collisions. Singly occupied lattice sites can be continuously imaged for several seconds. This method is suitable for high-resolution imaging of a many-body system in the Fermi-Hubbard regime.

  7. In situ Hybridization of Embryos with Antisense RNA Probes

    E-print Network

    Maduro, Morris F.

    Synthesis Template C. DIG-Labeled Probe Synthesis IV. Animal Preparation and Fixation A. Synchronization-Cracking and Fixation D. Hydration Series V. Hybridization and Signal Development A. Prehybridization and Probe

  8. Detection of Hepatitis B Virus DNA in Hepatocytes, Bile Duct Epithelium, and Vascular Elements by in situ Hybridization

    NASA Astrophysics Data System (ADS)

    Blum, Hubert E.; Stowring, Linda; Figus, Annalena; Montgomery, Carolyn K.; Haase, Ashley T.; Vyas, Girish N.

    1983-11-01

    A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefulness of in situ hybridization in the study of pathogenesis of HBV infection.

  9. Development of a combined portable x-ray fluorescence and Raman spectrometer for in situ analysis

    NASA Astrophysics Data System (ADS)

    Guerra, M.; Longelin, S.; Pessanha, S.; Manso, M.; Carvalho, M. L.

    2014-06-01

    In this work, we have built a portable X-ray fluorescence (XRF) spectrometer in a planar configuration coupled to a Raman head and a digital optical microscope, for in situ analysis. Several geometries for the XRF apparatus and digital microscope are possible in order to overcome spatial constraints and provide better measurement conditions. With this combined spectrometer, we are now able to perform XRF and Raman measurements in the same point without the need for sample collection, which can be crucial when dealing with cultural heritage objects, as well as forensic analysis. We show the capabilities of the spectrometer by measuring several standard reference materials, as well as other samples usually encountered in cultural heritage, geological, as well as biomedical studies.

  10. Use of X-ray fluorescence for in-situ detection of metals

    SciTech Connect

    Elam, W.T.; Whitlock, R.R.; Gilfrich, J.V.

    1995-12-31

    X-ray fluorescence (XRF) is a well-established, non-destructive method of determining elemental concentrations at ppm levels in complex samples. It can operate in atmosphere with no sample preparation, and provides accuracies of 1% or better under optimum conditions. This report addresses two sets of issues concerning the use of X-ray fluorescence as a sensor technology for the cone penetrometer, for shipboard waste disposal, or for other in-situ, real-time environmental applications. The first issue concerns the applicability of XRF to these applications, and includes investigation of detection limits and matrix effects. The authors have evaluated the detection limits and quantitative accuracy of a sensor mock-up for metals in soils under conditions expected in the field. In addition, several novel ways of improving the lower limits of detection to reach the drinking water regulatory limits have been explored. The second issue is the engineering involved with constructing a spectrometer within the 1.75 inch diameter of the penetrometer pipe, which is the most rigorous physical constraint. Only small improvements over current state-of-the-art are required. Additional advantages of XRF are that no radioactive sources or hazardous materials are used in the sensor design, and no reagents or any possible sources of ignition are involved.

  11. Novel, in-situ Raman and fluorescence measurement techniques: Imaging using optical waveguides

    NASA Astrophysics Data System (ADS)

    Carter, Jerry Chance

    The following dissertation describes the development of methods for performing standoff and in- situ Raman and fluorescence spectroscopy for chemical imaging and non-imaging analytical applications. The use of Raman spectroscopy for the in- situ identification of crack cocaine and cocaine.HCl using a fiberoptic Raman probe and a portable Raman spectrograph has been demonstrated. We show that the Raman spectra of both forms of cocaine are easily distinguishable from common cutting agents and impurities such as benzocaine and lidocaine. We have also demonstrated the use of Raman spectroscopy for in-situ identification of drugs separated by thin layer chromatography. We have investigated the use of small, transportable, Raman systems for standoff Raman spectroscopy (e.g. <20 m). For this work, acousto-optical (AOTF) and liquid crystal tunable filters (LCTF) are being used both with, and in place of dispersive spectrographs and fixed filtering devices. In addition, we improved the flexibility of the system by the use of a modified holographic fiber-optic probe for light and image collection. A comparison of tunable filter technologies for standoff Raman imaging is discussed along with the merits of image transfer devices using small diameter image guides. A standoff Raman imaging system has been developed that utilizes a unique polymer collection mirror. The techniques used to produce these mirrors make it easy to design low f/# polymer mirrors. The performance of a low f/# polymer mirror system for standoff Raman chemical imaging has been demonstrated and evaluated. We have also demonstrated remote Raman hyperspectral imaging using a dimension-reduction, 2-dimensional (2-D) to 1-dimensional (1-D), fiber optic array. In these studies, a modified holographic fiber-optic probe was combined with the dimension-reduction fiber array for remote Raman imaging. The utility of this setup for standoff Raman imaging is demonstrated by monitoring the polymerization of dibromostyrene. To further demonstrate the utility of in- situ spectral imaging, we have shown that small diameter (350 ?m) image guides can be used for in-situ measurements of analyte transport in thin membranes. This has been applied to the measurement of H2O diffusion in Nafion™ membranes using the luminescent compound, [Ru(phen)2dppz] 2+, which is a H2O indicator.

  12. Site Specific Synthesis and in-situ Immobilization of Fluorescent Silver Nanoclusters on DNA Nanoscaffolds Using Tollens Reaction

    SciTech Connect

    Pal, Suchetan; Varghese, R.; Yan, Hao; Liu, Yan

    2011-04-06

    DNA strands with specific sequences and covalently attached sugar moieties were used for the site-specific incorporation of the sugar units on a DNA origami scaffold. This approach enabled the subsequent site-specific synthesis and in situ immobilization of fluorescent Ag clusters at predefined positions on the DNA nanoscaffold by treatment with the Tollens reagent.

  13. Design of Hybrid Steam-In Situ Combustion Bitumen Recovery Processes

    SciTech Connect

    Yang Xiaomeng; Gates, Ian D.

    2009-09-15

    Given enormous capital costs, operating expenses, flue gas emissions, water treatment and handling costs of thermal in situ bitumen recovery processes, improving the overall efficiency by lowering energy requirements, environmental impact, and costs of these production techniques is a priority. Steam-assisted gravity drainage (SAGD) is the most widely used in situ recovery technique in Athabasca reservoirs. Steam generation is done on surface and consequently, because of heat losses, the energy efficiency of SAGD can never be ideal with respect to the energy delivered to the sandface. An alternative to surface steam generation is in situ combustion (ISC) where heat is generated within the formation through injection of oxygen at a sufficiently high pressure to initiate combustion of bitumen. In this manner, the heat from the combustion reactions can be used directly to mobilize the bitumen. As an alternative, the heat can be used to generate steam within the formation which then is the agent to move heat in the reservoir. In this research, alternative hybrid techniques with simultaneous and sequential steam-oxygen injection processes are examined to maximize the thermal efficiency of the recovery process. These hybrid processes have the advantage that during ISC, steam is generated within the reservoir from injected and formation water and as a product of oxidation. This implies that ex situ steam generation requirements are reduced and if there is in situ storage of combustion gases, that overall gas emissions are reduced. In this research, detailed reservoir simulations are done to examine the dynamics of hybrid processes to enable design of these processes. The results reveal that hybrid processes can lower emitted carbon dioxide-to-oil ratio by about 46%, decrease the consumed natural gas-to-oil ratio by about 73%, reduce the cumulative energy-to-oil ratio by between 40% and 70% compared to conventional SAGD, and drop water consumption per unit oil produced. However, oil recovery is between 25% and 40% below that of SAGD. Design of successful hybrid steam-oxygen processes must take into account the balance between injected steam and amount of injected oxygen and combustion gas products that dilute injected and in situ-generated steam in the depletion chamber by lowering its partial pressure, and thus its saturation temperature which in turn impacts production rates and recovery.

  14. [In situ hybridization in undifferentiated nasopharynx carcinoma in Tunisia. Report of 3 cases].

    PubMed

    Baizig Nehla, M; Hamouda, B; Asma, K; Farhat, B A; Abderahmen, L; Omrane, B H; Ahmed, el M

    2001-10-01

    The undifferentiated carcinoma of nasopharyngeal type (UCNT) is highly prevalent in South East Asia countries and has intermediate incidence in Tunisia, where Epstein-Barr virus constitutes one of the key factors of oncogenesis. Our preliminary results, in 3 patients with UCNT, show an elevated rate of EBV antibodies and a latent infection (type II) (expression of LMP and absence of ZEBRA by immunohistochemical reaction) and a positive staining with EBERs probe by in situ hybridation (ISH) in all cases. These results confirm a close association between EBV and tunisian UCNT. Moreover, the use of HIS technique for detection of EBERs constitutes an additional and formal argument of this association. PMID:11910697

  15. Quantitative methods for genome-scale analysis of in situ hybridization and correlation with microarray data

    PubMed Central

    Lee, Chang-Kyu; Sunkin, Susan M; Kuan, Chihchau; Thompson, Carol L; Pathak, Sayan; Ng, Lydia; Lau, Chris; Fischer, Shanna; Mortrud, Marty; Slaughterbeck, Cliff; Jones, Allan; Lein, Ed; Hawrylycz, Michael

    2008-01-01

    With the emergence of genome-wide colorimetric in situ hybridization (ISH) data sets such as the Allen Brain Atlas, it is important to understand the relationship between this gene expression modality and those derived from more quantitative based technologies. This study introduces a novel method for standardized relative quantification of colorimetric ISH signal that enables a large-scale cross-platform expression level comparison of ISH with two publicly available microarray brain data sources. PMID:18234097

  16. Hierarchically structured transparent hybrid membranes by in situ growth of mesostructured organosilica in host polymer

    NASA Astrophysics Data System (ADS)

    Vallé, Karine; Belleville, Philippe; Pereira, Franck; Sanchez, Clément

    2006-02-01

    The elaborate performances characterizing natural materials result from functional hierarchical constructions at scales ranging from nanometres to millimetres, each construction allowing the material to fit the physical or chemical demands occurring at these different levels. Hierarchically structured materials start to demonstrate a high input in numerous promising applied domains such as sensors, catalysis, optics, fuel cells, smart biologic and cosmetic vectors. In particular, hierarchical hybrid materials permit the accommodation of a maximum of elementary functions in a small volume, thereby optimizing complementary possibilities and properties between inorganic and organic components. The reported strategies combine sol-gel chemistry, self-assembly routes using templates that tune the material's architecture and texture with the use of larger inorganic, organic or biological templates such as latex, organogelator-derived fibres, nanolithographic techniques or controlled phase separation. We propose an approach to forming transparent hierarchical hybrid functionalized membranes using in situ generation of mesostructured hybrid phases inside a non-porogenic hydrophobic polymeric host matrix. We demonstrate that the control of the multiple affinities existing between organic and inorganic components allows us to design the length-scale partitioning of hybrid nanomaterials with tuned functionalities and desirable size organization from ångström to centimetre. After functionalization of the mesoporous hybrid silica component, the resulting membranes have good ionic conductivity offering interesting perspectives for the design of solid electrolytes, fuel cells and other ion-transport microdevices.

  17. Cytogenetic, interphase, and multicolor fluorescence in situ hybridization analyses in primary plasma cell leukemia: a study of 40 patients at diagnosis, on behalf of the Intergroupe Francophone du Myélome and the Groupe Français de Cytogénétique Hématologique.

    PubMed

    Avet-Loiseau, H; Daviet, A; Brigaudeau, C; Callet-Bauchu, E; Terré, C; Lafage-Pochitaloff, M; Désangles, F; Ramond, S; Talmant, P; Bataille, R

    2001-02-01

    Primary plasma cell leukemia (PCL) is a rare plasma cell malignancy. Consequently, few large reports have been published. Presented is a cytogenetic analysis of 40 patients with primary PCL compared with 247 newly diagnosed patients with stage III multiple myeloma (MM). Cytogenetic abnormalities were observed in 23 of 34 patients, with usually complex hypodiploid or pseudodiploid karyotypes. Analysis of rearrangements of the 14q32 region revealed significant differences with high cell mass MM-a higher incidence of t(11;14) (33% vs 16%; P <.025) and of t(14;16) (13% vs 1%; P <.002) though incidences of t(4;14) were identical and a higher incidence of monosomy 13 (68% vs 42%; P =.005). Hypodiploid karyotypes and monosomy 13 may explain, at least in part, the poorer prognosis of primary PCL. In contrast, significantly longer survival was observed in patients displaying t(11;14) in comparison with those lacking this translocation (P =.001). PMID:11157506

  18. Multicolor in situ hybridization and linkage analysis order Charcot-Marie-Tooth type I (CMTIA) gene-region markers

    SciTech Connect

    Lebo, R.V.; Lynch, E.D.; Golbus, M.S. ); Bird, T.D. ); Barker, D.F.; O'Connell, P.; Chance, P.F. )

    1992-01-01

    This study demonstrates a clear and current role for multicolor in situ hybridization in expediting positional cloning studies of unknown disease genes. Nine polymorphic DNA cosmids have been mapped to eight ordered locations spanning the Charcot-Marie-Tooth type 1 (CMT1A) disease gene region in distal band 17p11.2, by multicolor in situ hybridization. When used with linkage analysis, these methods have generated a fine physical map and have firmly assigned the CMT1A gene to distal band 17p11.2. Linkage analysis with four CMT1A pedigrees mapped the CMT1A gene with respect to two flanking markers. Additional loci were physically mapped and ordered by in situ hybridization and analysis of phase-known recombinants in CMT1A pedigrees. These data demonstrate the ability of in situ hybridization to resolve loci within 0.5 Mb on early-metaphase chromosomes. Multicolor in situ hybridization also excluded the possibility of pericentric inversions in two unrelated patients with CMT1 and neurofibromatosis type 1. When used with pulsed-field gel electrophoresis, multicolor in situ hybridization can establish physical location, order, and distance in closely spaced chromosome loci.

  19. Assignment of the phosducin (PDC) gene to human chromosome 1q25-1q32. 1 by somatic cell hybridization and in situ hybridization

    SciTech Connect

    Sparkes, R.S.; Kojis, T.; Klisak, I.; Heinzmann, C.; Bateman, J.B. ); Lee, R.H. ); Shinohara, T. ); Craft, C.M. )

    1993-11-01

    Phosducin is a soluble photoreceptor phosphoprotein that probably modulates phototransduction in the retina and thus qualifies as a potential candidate gene for retinitis pigmentosa. Using both human/mouse somatic cell hybrids and in situ hybridization to human metaphase chromosomes, the authors have mapped this gene to chromosome 1q25-1q32.1. 18 refs., 2 figs.

  20. An in situ antimicrobial susceptibility testing method based on in vivo measurements of chlorophyll ? fluorescence.

    PubMed

    Heliopoulos, Nikolaos S; Galeou, Angeliki; Papageorgiou, Sergios K; Favvas, Evangelos P; Katsaros, Fotios K; Stamatakis, Kostas

    2015-05-01

    Up to now antimicrobial susceptibility testing (AST) methods are indirect and generally involve the manual counting of bacterial colonies following the extraction of microorganisms from the surface under study and their inoculation in a separate procedure. In this work, an in situ, direct and instrumental method for the evaluation and assessment of antibacterial properties of materials and surfaces is proposed. Instead of indirectly determining antibacterial activity using the typical gram(-) test organisms with the subsequent manual colony count or inhibition zone measurement, the proposed procedure, employs photosynthetic gram(-) cyanobacteria deposited directly onto the surface under study and assesses cell proliferation and viability by a quick, accurate and reproducible instrumental chlorophyll fluorescence spectrophotometric technique. In contrast with existing methods of determination of antibacterial properties, it produces high resolution and quantitative results and is so versatile that it could be used to evaluate the antibacterial properties of any compound (organic, inorganic, natural or man-made) under any experimental conditions, depending on the targeted application. PMID:25771834

  1. Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans

    NASA Technical Reports Server (NTRS)

    Jurtshuk, R. J.; Blick, M.; Bresser, J.; Fox, G. E.; Jurtshuk, P. Jr

    1992-01-01

    A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.

  2. Using in situ hybridization and PFGE Southern hybridization to detect translocation breakpoints in a BOR/TRPS patient cell line

    SciTech Connect

    Gu, J.Z.; Sapru, M.; Smith, D.

    1994-09-01

    Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by ear malformations, cervical fistulae, hearing loss and renal abnormalities. We have integrated the Genethon YAC contig maps with additional markers in the chromosome 8q region genetically linked by a unique patient cell line. This cell line is from a patient who has both the branchio-oto-renal syndrome and tricho-rhino-phalangeal syndrome (TRPS). High resolution cytogenetics demonstrated a direct insertion of materials from 8q13.3q21.13 to 8q24.11. TRPS has been previously linked to deletions involving 8q24.11-q24.13. The rearrangement in this patient suggests that TRPS results from loss of gene function due to insertion at the 8q24.11 breakpoint and the possible location for the BOR gene is at either of the two breakpoints of 8q13.3 and 8q21.13. We have constructed cosmid contigs in 8q24.11. In situ hybridization with cosmids mapped to these locations as probes has helped to narrow down the breakpoints. Combinations of cosmids on either side or overlapping the 8q24.11 breakpoint show split signals on one chromosome 8q arm due to insertion of the materials from the proximal region. Cosmids mapped to the TRPS deletion region have been used to hybridize to pulsed field gel genomic blots of DNA from the patient cell line and detected rearranged genomic fragments. Both in situ hybridization and genomic PFGE Southern blot will be used to precisely locate the breakpoints.

  3. HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity.

    PubMed

    Hanna, Wedad M; Rüschoff, Josef; Bilous, Michael; Coudry, Renata A; Dowsett, Mitch; Osamura, Robert Y; Penault-Llorca, Frédérique; van de Vijver, Marc; Viale, Giuseppe

    2014-01-01

    Trastuzumab-containing therapy is a standard of care for patients with HER2+ breast cancer. HER2 status is routinely assigned using in situ hybridization to assess HER2 gene amplification, but interpretation of in situ hybridization results may be challenging in tumors with chromosome 17 polysomy or intratumoral genetic heterogeneity. Apparent chromosome 17 polysomy, defined by increased chromosome enumeration probe 17 (CEP17) signal number, is a common genetic aberration in breast cancer and represents an alternative mechanism for increasing HER2 copy number. Some studies have linked elevated CEP17 count ('polysomy') with adverse clinicopathologic features and HER2 overexpression, although there are numerous discrepancies in the literature. There is evidence that elevated CEP17 ('polysomy') count might account for trastuzumab response in tumors with normal HER2:CEP17 ratios. Nonetheless, recent studies establish that apparent 'polysomy' (CEP17 increase) is usually related to focal pericentromeric gains rather than true polysomy. Assigning HER2 status may also be complex where multiple cell subclones with distinct HER2 amplification characteristics coexist within the same tumor. Such genetic heterogeneity affects up to 40% of breast cancers when assessed according to a College of American Pathologists guideline, although other definitions have been proposed. Recent data have associated heterogeneity with unfavorable clinicopathologic variables and poor prognosis. Genetically heterogeneous tumors harboring HER2-amplified subclones have the potential to benefit from trastuzumab, but this has yet to be evaluated in clinical studies. In this review, we discuss the implications of apparent polysomy 17 and genetic heterogeneity for assigning HER2 status in clinical practice. Among our recommendations, we support the use of mean HER2 copy number rather than HER2:CEP17 ratio to define HER2 positivity in cases where coamplification of the centromere might mask HER2 amplification. We also highlight a need to harmonize in situ hybridization scoring methodology to support accurate HER2 status determination, particularly where there is evidence of heterogeneity. PMID:23807776

  4. In Situ Hybridization of Estrogen Receptors ? and ? and GPER in the Human Testis.

    PubMed

    Fietz, Daniela; Bergmann, M; Hartmann, K

    2016-01-01

    In situ hybridization (ISH) is an excellent method for detecting RNA in histological sections, both to detect gene expression and to assign gene expression to a distinct cell population. Therefore, ISH may be used in basic cell biology to detect the expression of certain genes within a tissue containing various cell populations. Here, we describe the detection and cellular localization of three estrogen receptors, both isoforms of the genomic estrogen receptor (ER? and ER?) as well as the membrane-bound G-protein-coupled estrogen receptor 1 (GPER) in the human testis. PMID:26585136

  5. Oligomeric interface modifiers in hybrid polymer solar cell prototypes investigated by fluorescence voltage spectroscopy.

    PubMed

    Reeja-Jayan, B; Koen, Katherine A; Ono, Robert J; Vanden Bout, David A; Bielawski, Christopher W; Manthiram, Arumugam

    2015-04-28

    Carboxylated oligothiophenes were evaluated as interfacial modifiers between the organic poly(3-hexylthiophene) (P3HT) and inorganic TiO2 layers in bilayer hybrid polymer solar cells. Carboxylated oligothiophenes can be isolated using conventional purification techniques resulting in pure, monodisperse molecules with 100% carboxylation. Device prototypes using carboxylated oligothiophenes as interfacial modifiers showed improved performance in the open-circuit voltage and fill factor over devices using unmodified oligothiophenes as interfacial modifiers. X-ray photoelectron spectroscopy (XPS) studies supported the idea that interface layer adhesion was improved by functionalizing oligothiophenes with a carboxyl moiety. Wide-field fluorescence images revealed that devices made using carboxylated oligothiophenes had fewer aggregates in the P3HT layers atop the modified TiO2 surface. Hysteresis seen in the fluorescence intensity as a function of applied bias, obtained from In-Device Fluorescence Voltage Spectroscopy (ID-FVS), was found to be a diagnostic criterion of the quality of the hybrid interface modification. The best interfaces were found using oligothiophenes functionalized with carboxylates, which created smooth layers on TiO2, and showed no hysteresis, suggesting elimination of interfacial charge traps. However, this hysteresis could be re-introduced by increasing the scan rate of the applied bias, suggesting that smooth P3HT layers created by carboxylated oligothiophene interface modifiers were necessary but not sufficient for sustaining improved photovoltaic properties especially during long-term device operation. PMID:25804286

  6. Assignment of the human RT6 gene to 11q13 by PCR screening of somatic cell hybrids and in situ hybridization

    SciTech Connect

    Koch-Nolte, F.; Haag, F.; Kuehl, M.; Thiele, H.G.; Singh, S. ); Van Heyningen, V. ); Hoovers, J. ); Grzeschik, K.H. )

    1993-11-01

    RT6 is a T cell membrane protein that has attracted interest because a defect in RT6 expression is associated with susceptibility to autoimmune type I diabetes in DP-BB rats and NOD mice. Using PCR screening of human/rodent somatic cell hybrids and fluorescence in situ hybridization, the authors have determined that the gene for the human RT6 homologue is located at 11q13, centromeric to the gene for tyrosinase (TYR, albino locus) and telomeric to that for fibroblast growth factor 4 (FGF4). The data suggest that the human RT6 gene constitutes a new linkage group with TYR and the gene for olfactory marker protein (OMP) on 11q, which has a counterpart in mouse chromosome 7. Thus, in the human, the RT6 locus is dissociated from the hemoglobin [beta] chain locus (HBB) and its neighboring conserved linkage group at 11q15, in contrast to the mouse, in which RT6 shows a tighter linkage to Hbb than to Tyr. The results support the conclusion that there has been considerable intrachromosomal reshuffling of linked genes since the divergence of primates and rodents. 9 refs., 1 fig.

  7. Tracking the composition and dominant components of the microbial community via polymerase chain reaction-denaturing gradient gel electrophoresis and fluorescence in situ hybridization during vermiconversion for liquid-state excess sludge stabilization.

    PubMed

    Xu, Ting; Xing, Meiyan; Yang, Jian; Lv, Baoyi; Duan, Ting; Nie, Jing

    2014-09-01

    To quantitatively explore the microbial community modified by earthworms, a vermifilter (VF, with earthworms) and a conventional biofilter (BF, without earthworms) were continuously operated to stabilize excess sludge. The results demonstrated a positive role imposed by earthworms on compositions and dominant components of microbial community in the VF. For one thing, the phyla Actinobacteria and Acidobacteria were only detected in the VF, which might explain for the higher Shannon index of bacteria in the VF (H = 2.58) than that in the BF (H = 1.99). For another, the total proportion of dominant bacteria in the VF increased by 23% compared to the BF. Moreover, quantification analysis explicitly noted that the dominant bacteria in VF were ?-proteobacteria (27 ± 2%) and ?-proteobacteria (24 ± 1%) while that in BF was Bacteroidetes (21 ± 1%). In conclusion, stimulated by earthworms, a unique microbial community developed in the VF, thus improving the stabilization of excess sludge. PMID:24971951

  8. Mapping of the receptor protein-tyrosine kinase 10 to human chromosome 1q21-q23 and mouse chromosome 1H1-5 by fluorescence in situ hybridization

    SciTech Connect

    Edelhoff, S.; Disteche, C.M.; Lai, C.

    1995-01-01

    Receptor protein-tyrosine kinases (PTKs) play a critical role in the transduction of signals important to cell growth, differentiation, and survival. Mutations affecting the expression of receptor PTK genes have been associated with a number of vertebrate and invertebrate developmental abnormalities, and the aberrant regulation of tyrosine phosphorylation is implicated in a variety of neoplasias. One estimate suggests that approximately 100 receptor PTK genes exist in the mammalian genome, about half of which have been identified. The tyro-10 receptor protein-tyrosine kinase, first identified in a PCR-based survey for novel tyrosine kinases in the rat nervous system, defines a new subfamily of PTKs. It exhibits a catalytic domain most closely related to those found in the trk PTK receptor subfamily, which transduces signals for nerve growth factor and the related molecules brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 (NT-3 and NT-4). Trk and the related PTK receptors trkB and trkC play a critical role in the neurotrophin-dependent survival of subsets of sensory and motor neurons. The predicted tyro-10 extracellular region is, however, distinct from that of the trk subfamily and is unique except for a domain shared with the blood coagulation factors V and VIII, thought to be involved in phospholipid binding. Although tyro-10 RNA is most abundant in heart and skeletal muscle in the adult rat, it is expressed in a wide variety of tissues, including the developing and mature brain. Tyro-10 appears identical to the murine TKT sequence reported by Karn et al. and exhibits a high degree of similarity with the CaK, DDR, and Nep PTKs. A ligand for tyro-10 has not yet been identified. 10 refs., 1 fig.

  9. Examination of equine glandular stomach lesions for bacteria, including Helicobacter spp by fluorescence in situ hybridisation

    PubMed Central

    2010-01-01

    Background The equine glandular stomach is commonly affected by erosion and ulceration. The aim of this study was to assess whether bacteria, including Helicobacter, could be involved in the aetiology of gastric glandular lesions seen in horses. Results Stomach lesions, as well as normal appearing mucosa were obtained from horses slaughtered for human consumption. All samples were tested for urease activity using the Pyloritek® assay, while mucosal bacterial content was evaluated using Fluorescence In Situ Hybridisation. In selected sub samples, bacteria characterisation was pursued further by cloning and sequencing. Mucosal lesions were found in 36/63 stomachs and included hyperplastic rugae, polypoid structures and focal erosions. None of the samples were tested positive for urease activity or for FISH using the Helicobacter genus specific probe. In samples of lesions, as well as normal samples, clones with 99% similarities to Lactobacillus salivarius and Sarcina ventriculi were found. Escherichia like bacterium clones and Enterococcus clones were demonstrated in one focal erosion. Based on a phylogenetic tree these clones had 100% similarity to Escherichia fergusonii and Enterococcus faecium. The Enterococcus were found colonising the mucosal surface, while E. fergusonii organisms were also demonstrated intraepithelial. Conclusion Gastric Helicobacter spp. could not be verified as being involved in lesions of the glandular stomach of the horse. Since E. fergusonii has been described as an emerging pathogen in both humans and animals, the finding of this bacterium in gastric erosion warrants further clarification to whether gastric infection with this type bacterium is important for horses. PMID:20298612

  10. Portable apparatus for in situ x-ray diffraction and fluorescence analyses of artworks.

    PubMed

    Eveno, Myriam; Moignard, Brice; Castaing, Jacques

    2011-10-01

    A portable X-ray fluorescence/X-ray diffraction (XRF/XRD) system for artwork studies has been designed constructed and tested. It is based on Debye Scherrer XRD in reflection that takes advantage of many recent improvements in the handling of X-rays (polycapillary optics; advanced two-dimensional detection). The apparatus is based on a copper anode air cooled X-ray source, and the XRD analysis is performed on a 5-20 ?m thick layer from the object surface. Energy dispersive XRF elemental analysis can be performed at the same point as XRD, giving elemental compositions that support the interpretation of XRD diagrams. XRF and XRD analyses were tested to explore the quality and the limits of the analytical technique. The XRD diagrams are comparable in quality with diagrams obtained with conventional laboratory equipment. The mineral identification of materials in artwork is routinely performed with the portable XRF-XRD system. Examples are given for ceramic glazes containing crystals and for paintings where the determination of pigments is still a challenge for nondestructive analysis. For instance, lead compounds that provide a variety of color pigments can be easily identified as well as a pigment such as lapis lazuli that is difficult to identify by XRF alone. More than 70 works of art have been studied in situ in museums, monuments, etc. In addition to ceramics and paintings, these works include bronzes, manuscripts, etc., which permit improvement in the comprehension of ancient artistic techniques. PMID:21615981

  11. A New In Situ Method of Determining Relative Abundances and Charge States of Implanted Transition Metals in Individual Grains Using Synchrotron X-Ray Fluorescence

    SciTech Connect

    Kitts, K.; Sutton, S.; Newville, M.

    2007-03-06

    We report on a new in situ method of determining relative abundances and charge states of implanted transition metals in individual grains using synchrotron X-ray fluorescence. In order to determine in situ the relative abundances and charge states of the transition metals in implanted solar wind in individual lunar plagioclase grains, we have developed a new microbeam x-ray fluorescence method using the synchrotron x-ray microprobe at the Advanced Photon Source (GSECARS sector 13) at Argonne National Laboratory.

  12. Mercury Vapor Release from Broken Compact Fluorescent Lamps and In Situ Capture by New Nanomaterial Sorbents

    PubMed Central

    2008-01-01

    The projected increase in the use of compact fluorescent lamps (CFLs) motivates the development of methods to manage consumer exposure to mercury and its environmental release at the end of lamp life. This work characterizes the time-resolved release of mercury vapor from broken CFLs and from underlying substrates after removal of glass fragments to simulate cleanup. In new lamps, mercury vapor is released gradually in amounts that reach 1.3 mg or 30% of the total lamp inventory after four days. Similar time profiles but smaller amounts are released from spent lamps or from underlying substrates. Nanoscale formulations of S, Se, Cu, Ni, Zn, Ag, and WS2 are evaluated for capture of Hg vapor under these conditions and compared to conventional microscale formulations. Adsorption capacities range over 7 orders of magnitude, from 0.005 (Zn micropowder) to 188?000 ?g/g (unstabilized nano-Se), depending on sorbent chemistry and particle size. Nanosynthesis offers clear advantages for most sorbent chemistries. Unstabilized nano-selenium in two forms (dry powder and impregnated cloth) was successfully used in a proof-of-principle test for the in situ, real-time suppression of Hg vapor escape following CFL fracture. PMID:18754507

  13. Automated microaxial tomography of cell nuclei after specific labelling by fluorescence in situ hybridisation.

    PubMed

    Kozubek, M; Skalníková, M; Matula, Pe; Bártová, E; Rauch, J; Neuhaus, F; Eipel, H; Hausmann, M

    2002-01-01

    Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre. PMID:12475562

  14. Diagnosis of Aujeszky's disease virus infection in dogs by use of immunohistochemistry and in-situ hybridization.

    PubMed

    Quiroga, M I; Nieto, J M; Sur, J; Osorio, F

    1998-03-01

    Aujeszky's disease (AD) was diagnosed in seven dogs by histological examination, immunohistochemistry and DNA in-situ hybridization. All dogs which lived on two swine farms died spontaneously showing an acute neurological syndrome (hypersalivation, vomiting, pruritus, depression and coma). The most significant histopathological lesion was a non-suppurative encephalitis located in the brain stem, mainly near the floor of the IVth ventricle. Aujeszky's disease virus (ADV) antigen and ADV nucleic acid distribution coincided with the histopathological lesions. However, little ADV antigen and ADV nucleic acid was found in severely damaged areas. A few neurons stained intensely by immunohistochemistry and in-situ hybridization in non-inflammatory areas. Both immunohistochemistry and in-situ hybridization techniques are valid techniques to confirm ADV infection in paraffin-embedded tissues and will be useful for characterizing the pathogenesis of ADV in the central nervous system (CNS) in carnivores. PMID:9591471

  15. Simple flow through reaction cells for in situ transmission and fluorescence x-ray-absorption spectroscopy of heterogeneous catalysts

    NASA Astrophysics Data System (ADS)

    Bare, Simon R.; Mickelson, George E.; Modica, Frank S.; Ringwelski, Andrzej Z.; Yang, N.

    2006-02-01

    We report on the design of both transmission and fluorescence x-ray-absorption spectroscopy cells suitable for in situ characterization of heterogeneous catalysts. The heart of both cells is a quartz tube used to house the catalyst sample. Both cells allow in situ x-ray-absorption fine-structure (XAFS) data to be recorded from -196 to 825 °C using a wide range of gas flows at atmospheric pressure. Excellent temperature control is demonstrated with both designs. XAFS data can be recorded over a wide x-ray energy range (2.1-29 keV). These designs are simple, robust, relatively low cost, and, moreover, are reliable and easy to operate. All of the critical components of the transmission reactor can be purchased commercially, with little machining required. The design of the fluorescence reactor requires access to a skilled glass blower.

  16. Making a Hybrid Microfluidic Platform Compatible for In Situ Imaging by Vacuum-Based Techniques

    SciTech Connect

    Yang, Li; Yu, Xiao-Ying; Zhu, Zihua; Thevuthasan, Suntharampillai; Cowin, James P.

    2011-10-26

    A self-contained microfluidic-based device was designed and fabricated for in situ imaging of aqueous surfaces using vacuum techniques. The device is a hybrid between a microfluidic PDMS block and external accessories, all portable on a small platform (10 cm-8 cm). The key feature is that a small aperture with a diameter of 2-3 micrometers is opened to the vacuum, which serves as a detection window for in situ imaging of aqueous surfaces. Vacuum compatibility and temperature drop due to water vaporization are the two most important challenges in this invention. Theoretical calculations and fabrication strategies are presented from multiple design aspects. In addition, results from the time-of-flight secondary ion mass spectrometry (ToF-SIMS) of aqueous surfaces are presented.

  17. Reconfigurable hybrid interface for molecular marker diagnostics and in-situ reporting.

    PubMed

    Ehrhardt, Kristina; Guinn, Michael T; Quarton, Tyler; Zhang, Michael Q; Bleris, Leonidas

    2015-12-15

    Combinations of molecular signals such as transcription factors and microRNAs in cells are a reliable indicator of multi-gene disorders. A system capable of detecting these conditions in-situ may be used as a tool for diagnosis and monitoring of disease. Here, we engineer genetic circuits that sense endogenous levels of the androgen receptor (AR), the glucocorticoid receptor (GR), and the microRNA hsa-miR-21 (miR-21) in cervical cancer cells (HeLa). Furthermore, using the mediator molecule human chorionic gonadotropin (hCG), we interface the intracellular information to enzyme-linked immunosorbent assay (ELISA) test strips. We demonstrate that this hybrid genetic circuit and test-strip interface can accommodate combinatorial, low-cost, and in-situ reporting, a versatile profiling tool. PMID:26210472

  18. A new application of click chemistry in situ: development of fluorescent probe for specific G-quadruplex topology.

    PubMed

    Hu, Ming-Hao; Chen, Xiao; Chen, Shuo-Bin; Ou, Tian-Miao; Yao, Meicun; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2015-01-01

    Target-guided synthesis is an approach to drug discovery that allows the target to self-assemble its own binding agents. So far, target-guided synthesis and especially in situ click chemistry have attracted extensive attention and have led to the identification of highly potent inhibitors for proteins. In this study, we expand the application of in situ click chemistry and present a procedure using this approach to identify selective fluorescent probes for a specific topology of G-quadruplex nucleic acids, the parallel G-quadruplexes. On this basis, compound 15 assembled by triarylimidazole scaffold and carboxyl side chain was a positive hit, demonstrating highly potential in the sensitive and selective detection of parallel G-quadruplexes. Such selective fluorescence response can be rationalized in terms of different binding affinities between 15 and G-quadruplexes. Our work accordingly represents a new development towards the application of in situ click chemistry to develop selective fluorescent probes and may also shed light on the search for probes for a specific G-quadruplex topology. PMID:26603780

  19. A new application of click chemistry in situ: development of fluorescent probe for specific G-quadruplex topology

    PubMed Central

    Hu, Ming-Hao; Chen, Xiao; Chen, Shuo-Bin; Ou, Tian-Miao; Yao, Meicun; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2015-01-01

    Target-guided synthesis is an approach to drug discovery that allows the target to self-assemble its own binding agents. So far, target-guided synthesis and especially in situ click chemistry have attracted extensive attention and have led to the identification of highly potent inhibitors for proteins. In this study, we expand the application of in situ click chemistry and present a procedure using this approach to identify selective fluorescent probes for a specific topology of G-quadruplex nucleic acids, the parallel G-quadruplexes. On this basis, compound 15 assembled by triarylimidazole scaffold and carboxyl side chain was a positive hit, demonstrating highly potential in the sensitive and selective detection of parallel G-quadruplexes. Such selective fluorescence response can be rationalized in terms of different binding affinities between 15 and G-quadruplexes. Our work accordingly represents a new development towards the application of in situ click chemistry to develop selective fluorescent probes and may also shed light on the search for probes for a specific G-quadruplex topology. PMID:26603780

  20. Localization of 3 beta-hydroxysteroid dehydrogenase in rat brain as studied by in situ hybridization.

    PubMed

    Dupont, E; Simard, J; Luu-The, V; Labrie, F; Pelletier, G

    1994-04-01

    Recent evidence suggests that brain cells can synthesize steroids de novo. The steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) gene family of enzymes is involved in the biosynthesis of all classes of active steroids. In the present study, we have used in situ hybridization performed with a 35S-labeled cDNA-encoding rat type I 3 beta-HSD to localize the expression of member(s) of this gene family in the adult rat brain. A specific hybridization signal could only be detected in a restricted area of the medulla ventrally and laterally bordering the fourth ventricle. This area contains the nucleus propositus hypoglossi as well as some vestibular nuclei: the nucleus vestibularis medialis, the nucleus vestibularis lateralis, and the nucleus vestibularis spinalis. At high resolution, the silver grains representing a hybridization signal were detected exclusively in neurons. The present data strongly suggest that a restricted population of neurons might be involved in steroid biosynthesis in the adult rat brain. The role of the member(s) of the rat 3 beta-HSD family in the nuclei of two cranial nerves remains to be investigated. PMID:8032681

  1. In Situ Hybridization Combined With Antibody Staining On Tissue Sections

    E-print Network

    Jessell, Thomas

    paraformaldehyde heat to 60-70 C, add 1 drop 10 N NaOH, and stir 10 minutes to dissolve add 50 ml 0.2M P.B. (final conc. 0.1M) sterile filter and store on ice up to 1 day 30% Sucrose in 0.1M P.B. solution 100 ml 0.2M P) and heat to 80 °C for 5'. Then transfer to ice for 5'. 11. Replace prehyb with 100ul heated/cooled probe

  2. In situ formation of fluorescent copper nanoparticles for ultrafast zero-background Cu(2+) detection and its toxicides screening.

    PubMed

    Qing, Zhihe; Zhu, Lixuan; Yang, Sheng; Cao, Zhong; He, Xiaoxiao; Wang, Kemin; Yang, Ronghua

    2016-04-15

    Copper pollution has become more and more serious in modern society as the increasing industrial emission and the acid mine drainage, and exposure to excess copper can result in damage to living organisms. Thus, the development of efficient strategy for copper ion (Cu(2+)) detection is very essential and significant. Here, a high-efficiency fluorescent method is proposed for Cu(2+) monitoring. The detection mechanism is based on the in situ formation of fluorescent copper nanoparticles (CuNPs). When the water sample is polluted by Cu(2+), fluorescence emission of CuNPs can be observed by a one-step manner, and the emission intensity is proportional to Cu(2+) concentration. Attractively, besides its advantages in operation and good detection capability, the generation of fluorescent signal is ultrafast, with a good signal response in 1min; and there is no interference from background and other ions due to the in situ formation of signal unit. By virtue of its advantages, this strategy has been used to detect Cu(2+) from polluted tap and river water samples, good performances demonstrate that the proposed method can be practically applied for Cu(2+) monitoring in real drinking and environmental water. Simultaneously, great potential for Cu(2+) toxicides screening has been verified by direct analysis of the effects of different model molecules on Cu(2+), which will contribute to Cu(2+)-related sewage treatment and medical therapy. PMID:26657590

  3. In Situ Airborne Measurement of Formaldehyde with a New Laser Induced Fluorescence Instrument

    NASA Astrophysics Data System (ADS)

    Arkinson, H.; Hanisco, T. F.; Cazorla, M.; Fried, A.; Walega, J.

    2012-12-01

    Formaldehyde (HCHO) is a highly reactive and ubiquitous compound in the atmosphere that originates from primary emissions and secondary formation by photochemical oxidation of volatile organic compounds. HCHO is an important precursor to the formation of ozone and an ideal tracer for the transport of boundary layer pollutants to higher altitudes. In situ measurements of HCHO are needed to improve understanding of convective transport mechanisms and the effects of lofted pollutants on ozone production and cloud microphysics in the upper troposphere. The Deep Convective Clouds and Chemistry Project (DC3) field campaign addressed the effects of deep, midlatitude continental convective clouds on the upper troposphere by examining vertical transport of fresh emissions and water aloft and by characterizing subsequent changes in composition and chemistry. Observations targeting convective storms were conducted over Colorado, Alabama, and Texas and Oklahoma. We present measurements of the In Situ Airborne Formaldehyde instrument (ISAF), which uses laser induced fluorescence to achieve the high sensitivity and fast time response required to detect low concentrations in the upper troposphere and capture the fine structure characteristic of convective storm outflow. Preliminary results from DC3 indicate that the ISAF is able to resolve concentrations ranging from under 35 ppt to over 35 ppb, spanning three orders of magnitude, in less than a few minutes. Frequent, abrupt changes in HCHO captured by the ISAF are corroborated by similar patterns observed by simultaneous trace gas and aerosol measurements. Primary HCHO emissions are apparent in cases when the DC-8 flew over combustion sources or biomass burning, and secondary HCHO formation is suggested by observations of enhanced HCHO concurrent with other elevated hydrocarbons. Vertical transport of HCHO is indicated by measurements of over 6 ppb from outflow in the upper troposphere. The DC-8 payload also included the Difference Frequency Generation Absorption Spectrometer instrument (DFGAS), which detects HCHO via infrared absorption. Preliminary data suggests the ISAF and DFGAS HCHO measurements typically agree to within 15%. Initial results from DC3 illustrate the efficacy of the novel ISAF instrument for detection of HCHO to elucidate convective transport of boundary layer pollutants and subsequent effects in the upper troposphere.

  4. In Situ Synthesis of Nanosized ZnO Particle/Organic Hybrids

    NASA Astrophysics Data System (ADS)

    Yogo, Toshinobu; Nakafuku, Tomoko; Kachi, Masahiro; Sakamoto, Wataru; Hirano, Shin?ichi

    2006-07-01

    A nanocrystalline ZnO particle/organic hybrid was synthesized in situ from zinc acrylate (ZA) using hydrazine derivatives. Nanosized ZnO particles were formed in the organic matrix by hydrolysis and polymerization of ZA at 65 °C. Methylhydrazine was the most suitable reagent for the synthesis of nanosized ZnO particles in the organic matrix. The crystallinity of ZnO particles depended upon the amount of water and the reaction time and increased with increasing reaction time as well as with the amount of water. Highly transparent ZnO particle/organic film was prepared from the hybrid solid between silica plates by pressing at 50 °C. UV-visible spectra showed an absorption edge corresponding to the size of the ZnO particles. The band gap energy of the hybrid was dependent upon the particle size, which reflected the synthesis conditions. ZnO particles in the hybrid exhibited stabilized particle size and absorption edge through chemical bonds to the polymer matrix at ambient temperature.

  5. A method for in situ hybridization in wholemounted lamprey brain: neurofilament expression in larvae and adults.

    PubMed

    Swain, G P; Jacobs, A J; Frei, E; Selzer, M E

    1994-04-01

    Nonisotopic in situ hybridization (NISH) using both cDNA and cRNA probes is rapidly gaining favor over autoradiographic methods. Typically, either biotinylated or digoxigenin-labeled probes are used to detect mRNAs in sectioned tissue or in cultured cells. With a few exceptions, most applications of NISH in wholemount preparations have been limited to Drosophila embryos. A protocol developed for NISH in whole adult Drosophila CNS was extended to wholemounted larval and adult lamprey brain preparations. Digoxigenin-labeled RNA probes were transcribed from cloned fragments of a lamprey neurofilament (NF180) cDNA. Hybridization with these probes, and comparisons with Nissl-stained wholemounts and wholemounts retrogradely labeled by injections of tracer into the spinal cord, demonstrated that NF180 mRNA was expressed in only a subset of neurons in the lamprey CNS. These included primarily neurons with long axons that project out of the brainstem, e.g., reticulospinal neurons and cranial motor neurons. Metamorphosis from the larval to the adult form was accompanied by an increase in the number of neurons expressing NF180 and in the apparent level of NF expression as judged by the intensity of labeling. For example, in the oculomotor and trochlear nuclei, expression of NF180 was seen in postmetamorphic young adult lampreys but not in larvae. In the trigeminal motor nucleus, both the number of neurons expressing NF180 and the intensity of the hybridization labeling increased with metamorphosis. The ability to do NISH in lamprey brain wholemounts eliminates the need for serial reconstructions and thus facilitates the study of selected gene expression during metamorphosis and regeneration. PMID:7523177

  6. Au nanoparticles enhanced fluorescence detection of DNA hybridization in picoliter microfluidic droplets.

    PubMed

    Zhu, Hongwei; Wang, Guodong; Xie, Donglei; Cai, Bo; Liu, Yumin; Zhao, Xingzhong

    2014-06-01

    This work reports a facile microfluidic device for Au-nanoparticle enhanced fluorescence detection of tiny amount of nucleotides within droplets in a high-throughput way. Droplets containing single strand DNA probe and relevant complementary strands DNA(cDNA) are generated in flow-focusing manner and the hybridization between them is realized in droplets flowing along a long serpentine channel. In order to find the optimal experimental condition, finite element method simulation is used to predict the interface evolution between the two phase liquids. Based on the fluorescence emited by intercalator reacted with the generated double-strand DNA(dsDNA), the target cDNA with a concentration of 1nM can be detected in droplets. And when we adopt Au nanoparticles to immobilize DNA probe which can amplify the fluorescence intensity, 10pM completary DNA could be detected. Due to the advantages in high-throughput and compartmentalization of this droplet platform, the detection procedure can be finished in 3 h. Our method shows good potential application in facile, sensitive, low cost and fast DNA detection for applications in personal health care. PMID:24599582

  7. Cellular localization of nerve growth factor synthesis by in situ hybridization.

    PubMed Central

    Bandtlow, C E; Heumann, R; Schwab, M E; Thoenen, H

    1987-01-01

    A very sensitive and specific method for in situ hybridization has been developed. This method detects low copy numbers of mRNA(NGF) transcripts in both tissue sections and cultured cells using 35S-labelled cRNA and oligonucleotide probes. In order to reduce the high nonspecific background occurring with 35S-labelled probes, prehybridization in the presence of non-labelled thio alpha UTP at pH 5.5 proved to be essential, together with a series of additional changes in the standard procedures for in situ hybridization. With this improved method it was possible to demonstrate that in tissues densely innervated by sensory (whisker pad) or both sympathetic and sensory (iris) fibers, NGF is synthesized not only by Schwann cells ensheathing these fibers, but also--and even to a much larger extent--by the target cells of the sensory and sympathetic neurons, i.e. epithelial cells, smooth muscle cells and fibroblasts. Moreover, in the sciatic nerve of newborn rats (where the mRNA(NGF) levels are 15 X higher than in adults) it was demonstrated that all Schwann cells have the capacity to express mRNA(NGF), not just those ensheathing the axons of NGF-responsive neurons. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3595562

  8. In situ hybridization: use of 35S-labeled probes on paraffin tissue sections.

    PubMed

    Micales, B K; Lyons, G E

    2001-04-01

    The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided. PMID:11316432

  9. Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

    SciTech Connect

    Deutsch, Eric W.; Ball, Catherine A.; Berman, Jules J.; Bova, G. Steven; Brazma, Alvis; Bumgarner, Roger E.; Campbell, David; Causton, Helen C.; Christiansen, Jeff; Daian, Fabrice; Dauga, Delphine; Davidson, Duncan; Gimenez, Gregory; Goo, Young Ah; Grimmond, Sean; Henrich, Thorsten; Herrmann, Bernhard G.; Johnson, Michael H.; Korb, Martin; Mills, Jason C.; Oudes, Asa; Parkinson, Helen E.; Pascal, Laura E.; Pollet, Nicolas; Quackenbush, John; Ramaialison, Mirana; Ringwald, Martin; Salgado, David; Sansone, Susanna A.; Sherlock, Gavin; Stoeckert, Christian Jr. J.; Swedlow, Jason; Taylor, Ronald C.; Walasheck, Laura; Warford, Anthony; Wilkinson, David G.; Zhou, Yi; Zon, Leonard I.; Liu, Alvin Y.; True, Lawrence D.

    2008-03-28

    Herein, we present for consideration such a specification, termed “Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)”. It is modelled after the MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments. The purpose of data standards like MIAME and MISFISHIE is to specify information content without specifying a format for encoding that information. The MISFISHIE standard specifies six sections of information that must be detailed for each experiment: Experimental Design, Specimens, Reporters, Staining, Imaging Data, and Image Characterizations. A general checklist is provided to quickly and efficiently establish adherence to the standard. Currently, we estimate that most articles describing gene expression localization studies, such as in situ hybridization assays, do not fully provide the minimum information needed for independent verification of results. In a small survey of 32 journal articles from the past five years, we found that nearly 90% did not meet all the requirements, although many met most of them. We propose that requiring authors to provide the minimum experimental detail about gene expression localization experiments would substantially facilitate reproducibility and interpretability of results by fellow investigators. Furthermore, inclusion of specific experimental details such as reagents and methods in publications would ultimately allow others to readily search the literature for these data items, especially given the ongoing trend towards open access full text journals.

  10. X-Ray Diffraction and Fluorescence Measurements for In Situ Planetary Instruments

    NASA Astrophysics Data System (ADS)

    Hansford, G.; Hill, K. S.; Talboys, D.; Vernon, D.; Ambrosi, R.; Bridges, J.; Hutchinson, I.; Marinangeli, L.

    2011-12-01

    The ESA/NASA ExoMars mission, due for launch in 2018, has a combined X-ray fluorescence/diffraction instrument, Mars-XRD, as part of the onboard analytical laboratory. The results of some XRF (X-ray fluorescence) and XRD (X-ray diffraction) tests using a laboratory chamber with representative performance are reported. A range of standard geological reference materials and analogues were used in these tests. The XRD instruments are core components of the forthcoming NASA Mars Science Laboratory (MSL) and ESA/NASA ExoMars missions and will provide the first demonstrations of the capabilities of combined XRD/XRF instrumentation in situ on an extraterrestrial planetary surface. The University of Leicester team is part of the Italy-UK collaboration that is responsible for building the ExoMars X-ray diffraction instrument, Mars-XRD [1,2]. Mars-XRD incorporates an Fe-55 radioisotope source and three fixed-position charge-coupled devices (CCDs) to simultaneously acquire an X-ray fluorescence spectrum and a diffraction pattern providing a measurement of both elemental and mineralogical composition. The CCDs cover an angular range of 2? = 6° to 73° enabling the analysis of a wide range of geologically important minerals including phyllosilicates, feldspars, oxides, carbonates and evaporites. The identification of hydrous minerals may help identify past Martian hydrothermal systems capable of preserving traces of life. Here we present some initial findings from XRF and XRD tests carried out at the University of Leicester using an Fe-55 source and X-ray sensitive CCD. The XRF/XRD test system consists of a single CCD on a motorised arm, an Fe-55 X-ray source, a collimator and a sample table which approximately replicate the reflection geometry of the Mars-XRD instrument. It was used to test geological reference standard materials and Martian analogues. This work was funded by the Science and Technology Facilities Council, UK. References [1] Marinangeli, L., Hutchinson, I., Baliva, A., Stevoli, A., Ambrosi, R., Critani, F., Delhez, R., Scandelli, L., Holland, A., Nelms, N. & the Mars-XRD Team, Proceedings of the 38th Lunar and Planetary Science Conference, 12 - 16 March 2007, League City, Texas, USA. [2] L. Marinangeli, I. B. Hutchinson, A. Stevoli, G. Adami, R. Ambrosi, R. Amils, V. Assis Fernandes, A. Baliva, A. T. Basilevsky, G. Benedix, P. Bland, A. J. Böttger, J. Bridges, G. Caprarelli, G. Cressey, F. Critani, N. d'Alessandro, R. Delhez, C. Domeneghetti, D. Fernandez-Remolar, R. Filippone, A. M. Fioretti, J. M. Garcia Ruiz, M. Gilmore, G. M. Hansford, G. Iezzi, R. Ingley, M. Ivanov, G. Marseguerra, L. Moroz, C. Pelliciari, P. Petrinca, E. Piluso, L. Pompilio, J. Sykes, F. Westall and the MARS-XRD Team, EPSC-DPS Joint Meeting 2011, 3 - 7 October 2011, La Cité Internationale des Congrès Nantes Métropole, Nantes, France.

  11. Resolution-improved in situ DNA hybridization detection based on microwave photonic interrogation.

    PubMed

    Cao, Yuan; Guo, Tuan; Wang, Xudong; Sun, Dandan; Ran, Yang; Feng, Xinhuan; Guan, Bai-Ou

    2015-10-19

    In situ bio-sensing system based on microwave photonics filter (MPF) interrogation method with improved resolution is proposed and experimentally demonstrated. A microfiber Bragg grating (mFBG) is used as sensing probe for DNA hybridization detection. Different from the traditional wavelength monitoring technique, we use the frequency interrogation scheme for resolution-improved bio-sensing detection. Experimental results show that the frequency shift of MPF notch presents a linear response to the surrounding refractive index (SRI) change over the range of 1.33 to 1.38, with a SRI resolution up to 2.6 × 10-5 RIU, which has been increased for almost two orders of magnitude compared with the traditional fundamental mode monitoring technique (~3.6 × 10-3 RIU). Due to the high Q value (about 27), the whole process of DNA hybridization can be in situ monitored. The proposed MPF-based bio-sensing system provides a new interrogation method over the frequency domain with improved sensing resolution and rapid interrogation rate for biochemical and environmental measurement. PMID:26480367

  12. SUPERSENSITIVE IN SITU HYBRIDIZATION BY TYRAMIDE SIGNAL AMPLIFICATION AND NANOGOLD SILVER STAINING: THE CONTRIBUTION OF AUTOMETALLOGRAPHY AND CATALYZED REPORTER DEPOSITION TO THE REJUVENATION OF IN SITU HYBRIDIZATION.

    SciTech Connect

    TUBBS,R.R.PETTAY,J.GROGAN,T.CHEUNG,A.L.M.POWELL,R.D.HAINFELD,J.HAUSER-KRONBERGER,C.HACKER,G.W.

    2002-04-17

    It is peculiar that in situ hybridization (ISH), a technique with many similarities to immunohistochemistry (IHC), has not enjoyed the phenomenal growth in both basic research and clinical applications as has its sister technique IHC. Since the late 1970s, when immunoperoxidase techniques began to be applied to routine diagnostic material and to numerous research applications, there has been a natural evolution of the IHC procedure. Namely, only a few primary antibodies were available commercially at the onset, and only one indirect and the peroxidase-antiperoxidase (PAP) technique detection systems were in place. With the advent of avidin-biotin detection systems and monoclonal antibodies, and a viable commercial market, extraordinary growth of the procedure's applications in clinical research and diagnostic pathology occurred during the subsequent two decades. Today, IHC is automated and widely used for research purposes and, to a large extent, has become a routine diagnostic ''special stain'' in most clinical laboratories. During the same period, ISH enjoyed very little growth in both research and diagnostic applications. What has accounted for this lack of maturation of the technique? The success of IHC is part of the reason measuring a gene's encoded protein routinely and inexpensively, particularly as automation evolved, rendered IHC a more viable choice in many instances. Inherent comparative sensitivity of the procedures has also clearly been a factor. Unfortunately, the chromogenic procedures in place are often insufficiently sensitive to detect the relatively low amounts of DNA and RNA levels at which the clinical utility is to be found.

  13. An in situ hybridization technique for the study of B19 human parvovirus replication in bone marrow cell cultures.

    PubMed

    Vassias, I; Perol, S; Coulombel, L; Thebault, M C; Lagrange, P H; Morinet, F

    1993-10-01

    An in situ hybridization technique using digoxigenin labelling was developed to study B19 infection. By using appropriate DNA probes, transcription of structural and non-structural genes was detected in bone marrow cell cultures. Such a simple system is useful to the study of B19-cell interactions in non-permissive cell lines. PMID:8263124

  14. Detection of high-risk human papillomavirus subtypes in cervical glandular neoplasia by in situ hybridization

    PubMed Central

    Sheng, Zhang; Minato, Hiroshi; Sasagawa, Toshiyuki; Nakada, Satoko; Kinoshita, Eriko; Kurose, Nozomu; Nojima, Takayuki; Makinoda, Satoru

    2013-01-01

    In situ hybridization (ISH) was performed on paraffin-embedded tissues to detect multiple high-risk human papillomavirus (HPV) subtypes in 27 cases of cervical adenocarcinoma in situ (AIS) and adenocarcinoma (CA) specimens. These results were compared with those of HPV detection by HPV-PCR genotyping and p16 immunohistochemistry in the same specimens. Of the 27 cases, 17 (63%) showed HPV-DNA by HPV-ISH, including 3 metastatic lesions. HPV-DNA was detected in 18 cases (67%) by PCR. The concordance rate between HPV-ISH and HPV-PCR genotyping was 74% with moderate agreement (Kappa value, 0.41). HPV-16 was identified in 5 cases, HPV-18 in 2 cases, and HPV-45 in 1 case. Combining the results of HPV-ISH and HPV-PCR/genotyping, 22 cases (81.5%) were considered HPV positive. Immunohistochemical staining of p16 indicated that 25 (93%) cases were positive; however, 4 of these cases were HPV-negative by both PCR and ISH. Combining HPV-ISH and HPV-PCR/genotyping techniques demonstrated a high sensitivity of HPV detection in FFPE tissues from cervical glandular neoplasias. In contrast, p16 immunohistochemistry seemed to have a low specificity for determining HPV status in cervical glandular neoplasia. HPV-ISH is useful for recognizing the distribution of HPV in AIS and CA tissues and visualizing signal patterns, and may be a useful tool to confirm the cervical origin of neoplasias and metastatic lesions. PMID:24133595

  15. Detection of high-risk human papillomavirus subtypes in cervical glandular neoplasia by in situ hybridization.

    PubMed

    Sheng, Zhang; Minato, Hiroshi; Sasagawa, Toshiyuki; Nakada, Satoko; Kinoshita, Eriko; Kurose, Nozomu; Nojima, Takayuki; Makinoda, Satoru

    2013-01-01

    In situ hybridization (ISH) was performed on paraffin-embedded tissues to detect multiple high-risk human papillomavirus (HPV) subtypes in 27 cases of cervical adenocarcinoma in situ (AIS) and adenocarcinoma (CA) specimens. These results were compared with those of HPV detection by HPV-PCR genotyping and p16 immunohistochemistry in the same specimens. Of the 27 cases, 17 (63%) showed HPV-DNA by HPV-ISH, including 3 metastatic lesions. HPV-DNA was detected in 18 cases (67%) by PCR. The concordance rate between HPV-ISH and HPV-PCR genotyping was 74% with moderate agreement (Kappa value, 0.41). HPV-16 was identified in 5 cases, HPV-18 in 2 cases, and HPV-45 in 1 case. Combining the results of HPV-ISH and HPV-PCR/genotyping, 22 cases (81.5%) were considered HPV positive. Immunohistochemical staining of p16 indicated that 25 (93%) cases were positive; however, 4 of these cases were HPV-negative by both PCR and ISH. Combining HPV-ISH and HPV-PCR/genotyping techniques demonstrated a high sensitivity of HPV detection in FFPE tissues from cervical glandular neoplasias. In contrast, p16 immunohistochemistry seemed to have a low specificity for determining HPV status in cervical glandular neoplasia. HPV-ISH is useful for recognizing the distribution of HPV in AIS and CA tissues and visualizing signal patterns, and may be a useful tool to confirm the cervical origin of neoplasias and metastatic lesions. PMID:24133595

  16. Nonradioactive in situ hybridization for detection of hydrophobin mRNA in the phytopathogenic fungus Claviceps purpurea during infection of rye.

    PubMed

    Tenberge, K B; Stellamanns, P; Plenz, G; Robenek, H

    1998-03-01

    Hydrophobins are unique fungal extracellular proteins that produce amphipathic films at interfaces, mediate contact to hydrophobic surfaces and are known to be important in phytopathogenicity. In the pathogenic ascomycete Claviceps purpurea, causing ergot disease in grasses and cereals and ergotism in livestock, a gene encoding an extraordinary type of hydrophobin has been detected, which appeared to be induced during alkaloid synthesis in axenic culture of an ergot-alkaloid producing strain of Claviceps (V. Garre and P. Tudzynski, pers. communication; Arntz and Tudzynski, 1997, Curr. Genet. 31, 357-360). To elucidate presence and function of this hydrophobin during infection of rye, the nonradioactive in situ hybridization technique was successfully adapted to the fungal organism and optimized in the pathogenic interaction system. Semithin cryosections proved to be suitable for microscopical gene expression analysis using immune-mediated alkaline-phosphatase staining for detection of digoxigenin-labeled cRNA probes. Specific hybridization of the prepared antisense riboprobe to hydrophobin mRNA was confirmed in nonradioactive Northern blots. While permeabilization by proteinase K had only a minor effect, the inclusion of detergent into the hybridization solutions enhanced specific RNA-RNA hybridization under maximum stringency. Hydrophobin mRNA was found in fungal cells, growing in axenic culture. In the disease cycle, hydrophobin transcripts were localized in abundance during vegetative fructification in conidiophores that actively produced conidia. No signals were observed in sclerotial hyphae during formation of the alkaloid-containing ergots, although they fluoresced intensely during total RNA detection using acridine orange. Notably, in situ hybridization experiments resulted in specific signals during early infection and colonization phases in the external mycelia and in hyphae penetrating the host epidermal layer. The presumed role of the hydrophobin gene product in ergot pathogenicity is discussed with respect to the described spatio-temporal distribution of the hydrophobin transcripts. PMID:9587058

  17. Localization of the expression of complement component 3 in the human endometrium by in situ hybridization

    SciTech Connect

    Sayegh, R.A.; Tao, Xiao Jing; Awwad, J.T.

    1996-04-01

    C3 production by the human endometrium has been previously described. The objective of the current study was to localize the site of expression and regulation of the third component of complement, C3, in the endometrium. Eight secretory and eight proliferative archival endometrial samples from hysterectomy and endometrial biopsy specimens were used for in situ hybridization analysis. This analysis was performed with a radiolabeled riboprobe synthesized from a 736-bp template representing sequence 1944-2680 of the human C3 complementary DNA. Duplicate sections were hybridized with sense and antisense riboprobes. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy. In proliferative endometrium, minimal expression of C3 was observed and was limited to a few stromal patches and glands throughout the section. In the secretory samples, prominent C3 expression was observed in both the glands and stroma of the basalis layer. Endometrial lymphocytes did not express C3. Endometrial stromal and glandular cells express the C3 gene. Endometrial lymphocytes did not express C3, but other nondistinct lymphoid elements scattered in the stroma may be expressing C3. There was a visibly more intense expression of C3 in the basalis layer of the secretory endometrium than in proliferative endometrium. The spatial and temporal pattern of C3 expression may have implications in normal menstrual physiology and in the immunological response of the endometrium to the invading trophoblast during placentation. 23 refs., 4 figs., 1 tab.

  18. In vivo self-bio-imaging of tumors through in situ biosynthesized fluorescent gold nanoclusters

    NASA Astrophysics Data System (ADS)

    Wang, Jianling; Zhang, Gen; Li, Qiwei; Jiang, Hui; Liu, Chongyang; Amatore, Christian; Wang, Xuemei

    2013-01-01

    Fluorescence imaging in vivo allows non-invasive tumor diagnostic thus permitting a direct monitoring of cancer therapies progresses. It is established herein that fluorescent gold nanoclusters are spontaneously biosynthesized by cancerous cell (i.e., HepG2, human hepatocarcinoma cell line; K562, leukemia cell line) incubated with micromolar chloroauric acid solutions, a biocompatible molecular Au(III) species. Gold nanoparticles form by Au(III) reduction inside cells cytoplasms and ultimately concentrate around their nucleoli, thus affording precise cell imaging. Importantly, this does not occur in non-cancerous cells, as evidenced with human embryo liver cells (L02) used as controls. This dichotomy is exploited for a new strategy for in vivo self-bio-imaging of tumors. Subcutaneous injections of millimolar chloroauric acid solution near xenograft tumors of the nude mouse model of hepatocellular carcinoma or chronic myeloid leukemia led to efficient biosynthesis of fluorescent gold nanoclusters without significant dissemination to the surrounding normal tissues, hence allowing specific fluorescent self-bio-marking of the tumors.

  19. Development of an in situ loop-mediated isothermal amplification technique for chromosomal localization of DNA sequences

    NASA Astrophysics Data System (ADS)

    Meng, Qinglei; Wang, Shi; Zhang, Lingling; Huang, Xiaoting; Bao, Zhenmin

    2013-01-01

    In situ loop-mediated isothermal amplification (in situ LAMP) combines in situ hybridization and loop-mediated isothermal amplification (LAMP) techniques for chromosomal localization of DNA sequences. In situ LAMP is a method that is generally more specific and sensitive than conventional techniques such as fluorescence in situ hybridization (FISH), primed in situ labeling (PRINS), and cycling primed in situ labeling (C-PRINS). Here, we describe the development and application of in situ LAMP to identify the chromosomal localization of DNA sequences. To benchmark this technique, we successfully applied this technique to localize the major ribosomal RNA gene on the chromosomes of the Zhikong scallop ( Chlamys farreri).

  20. Europium chelate labels in time-resolved fluorescence immunoassays and DNA hybridization assays

    SciTech Connect

    Diamandis, E.P.; Christopoulos, T.K. Univ. of Toronto, Ontario )

    1990-11-15

    Like many analytical methodologies, immunoassays and nucleic acid hybridization assays rely on the reaction between an analyte of interest and a specific reagent. The analyte concentration is then deduced by measuring either the amount of analyte-reagent complex formed (product) or the amount of residual reagent. The authors describe the application of fluorescent rare-earth chelates to immunoassay and DNA probing.

  1. In situ photochemical synthesis of fluorescent carbon dots for optical sensing of hydrogen peroxide and antioxidants.

    PubMed

    Costas-Mora, Isabel; Romero, Vanesa; Lavilla, Isela; Bendicho, Carlos

    2015-11-01

    A new synthesis approach for obtaining fluorescent carbon dots (CDs) based on UV irradiation of carbohydrates was developed. The photochemical synthesis pathway allows the formation of water soluble CDs of analytical usefulness within one min. CDs obtained by photochemical treatment from the sucrose/NaOH/poly(ethylene glycol) system are monodisperse with an average size of 8nm as determined by transmission electron microscopy. A dramatic increase in the CDs fluorescence (turn on) is observed when H2O2 is added. The decrease in CDs size occurring by the action of highly oxidant OH radicals gives rise to confinement of emissive energy traps and, in turn, to fluorescence enhancement. Antioxidants such as ascorbic acid and glutathione inhibit the photochemical reaction giving rise to a decrease in fluorescence of the CDs/H2O2 system (turn on-off). The detection limit was 5µM H2O2 and the repeatability expressed as the relative standard deviation was 3.8% (N=7). The photochemical synthesis of CDs allows building a green, low-cost, safe and fast assay for the detection of H2O2 and antioxidants. An application of the novel fluorescent nanoprobe to H2O2 detection in contact lens cleaning solutions is performed. PMID:26452963

  2. Localization during development of alternatively spliced forms of cytotactin mRNA by in situ hybridization

    PubMed Central

    1990-01-01

    Cytotactin, an extracellular glycoprotein found in neural and nonneural tissues, influences a variety of cellular phenomena, particularly cell adhesion and cell migration. Northern and Western blot analysis and in situ hybridization were used to determine localization of alternatively spliced forms of cytotactin in neural and nonneural tissues using a probe (CT) that detected all forms of cytotactin mRNA, and one (VbVc) that detected two of the differentially spliced repeats homologous to the type III repeats of fibronectin. In the brain, the levels of mRNA and protein increased from E8 through E15 and then gradually decreased until they were barely detectable by P3. Among the three cytotactin mRNAs (7.2, 6.6, and 6.4 kb) detected in the brain, the VbVc probe hybridized only to the 7.2-kb message. In isolated cerebella, the 220- kD polypeptide and 7.2-kb mRNA were the only cytotactin species present at hatching, indicating that the 220-kD polypeptide is encoded by the 7.2-kb message that contains the VbVc alternatively spliced insert. In situ hybridization showed cytotactin mRNA in glia and glial precursors in the ventricular zone throughout the central nervous system. In all regions of the nervous system, cytotactin mRNAs were more transient and more localized than the polypeptides. For example, in the radial glia, cytotactin mRNA was observed in the soma whereas the protein was present externally along the glial fibers. In the telencephalon, cytotactin mRNAs were found in a narrow band at the edge of a larger region in which the protein was wide-spread. Hybridization with the VbVc probe generally overlapped that of the CT probe in the spinal cord and cerebellum, consistent with the results of Northern blot analysis. In contrast, in the outermost tectal layers, differential hybridization was observed with the two probes. In nonneural tissues, hybridization with the CT probe, but not the VbVc probe, was detected in chondroblasts, tendinous tissues, and certain mesenchymal cells in the lung. In contrast, hybridization with both probes was observed in smooth muscle and lung epithelium. Both epithelium and mesenchyme expressed cytotactin mRNA in varying combinations: in the choroid plexus, only epithelial cells expressed cytotactin mRNA; in kidney, only mesenchymal cells; and in the lung, both of these cell types contained cytotactin mRNA. These spatiotemporal changes during development suggest that the synthesis of the various alternatively spliced cytotactin mRNAs is responsive to tissue-specific local signals and prompt a search for functional differences in the various molecular forms of the protein. PMID:1696267

  3. In situ preparation of fluorescent CdTe quantum dots with small thiols and hyperbranched polymers as co-stabilizers

    PubMed Central

    2014-01-01

    A new strategy for in situ preparation of highly fluorescent CdTe quantum dots (QDs) with 3-mercaptopropionic acid (MPA) and hyperbranched poly(amidoamine)s (HPAMAM) as co-stabilizers was proposed in this paper. MPA and HPAMAM were added in turn to coordinate Cd2+. After adding NaHTe and further microwave irradiation, fluorescent CdTe QDs stabilized by MPA and HPAMAM were obtained. Such a strategy avoids the aftertreatment of thiol-stabilized QDs in their bioapplication and provides an opportunity for direct biomedical use of QDs due to the existence of biocompatible HPAMAM. The resulting CdTe QDs combine the mechanical, biocompatibility properties of HPAMAM and the optical, electrical properties of CdTe QDs together. PMID:24636234

  4. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques.

    PubMed

    Wagner, D; Börgardts, M; Grünzweig, C; Lehmann, E; Müller, T J J; Egelhaaf, S U; Hermes, H E

    2015-09-01

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied. PMID:26429447

  5. Neutron, fluorescence, and optical imaging: An in situ combination of complementary techniques

    NASA Astrophysics Data System (ADS)

    Wagner, D.; Börgardts, M.; Grünzweig, C.; Lehmann, E.; Müller, T. J. J.; Egelhaaf, S. U.; Hermes, H. E.

    2015-09-01

    An apparatus which enables the simultaneous combination of three complementary imaging techniques, optical imaging, fluorescence imaging, and neutron radiography, is presented. While each individual technique can provide information on certain aspects of the sample and their time evolution, a combination of the three techniques in one setup provides a more complete and consistent data set. The setup can be used in transmission and reflection modes and thus with optically transparent as well as opaque samples. Its capabilities are illustrated with two examples. A polymer hydrogel represents a transparent sample and the diffusion of fluorescent particles into and through this polymer matrix is followed. In reflection mode, the absorption of solvent by a nile red-functionalized mesoporous silica powder and the corresponding change in fluorescent signal are studied.

  6. Airborne in-situ spectral characterization and concentration estimates of fluorescent organics as a function of depth

    NASA Technical Reports Server (NTRS)

    Tittle, R. A.

    1988-01-01

    The primary purpose of many in-situ airborne light scattering experiments in natural waters is to spectrally characterize the subsurface fluorescent organics and estimate their relative concentrations. This is often done by shining a laser beam into the water and monitoring its subsurface return signal. To do this with the proper interpretation, depth must be taken into account. If one disregards depth dependence when taking such estimates, both their spectral characteristics and their concentrations estimates can be rather ambiguous. A simple airborne lidar configuration is used to detect the subsurface return signal from a particular depth and wavelength. Underwater scatterometer were employed to show that in-situ subsurface organics are very sensitive to depth, but they also require the use of slow moving boats to cover large sample areas. Also, their very entry into the water disturbs the sample it is measuring. The method described is superior and simplest to any employed thus far.

  7. Improving the Detection Limit in a Capillary Raman System for In Situ Gas Analysis by Means of Fluorescence Reduction

    PubMed Central

    Rupp, Simone; Off, Andreas; Seitz-Moskaliuk, Hendrik; James, Timothy M.; Telle, Helmut H.

    2015-01-01

    Raman spectroscopy for low-pressure or trace gas analysis is rather challenging, in particular in process control applications requiring trace detection and real-time response; in general, enhancement techniques are required. One possible enhancement approach which enjoys increasing popularity makes use of an internally-reflective capillary as the gas cell. However, in the majority of cases, such capillary systems were often limited in their achievable sensitivity by a significant fluorescence background, which is generated as a consequence of interactions between the laser light and optical glass components in the setup. In order to understand and counteract these problems we have investigated a range of fluorescence-reducing measures, including the rearrangement of optical elements, and the replacement of glass components—including the capillary itself—by metal alternatives. These studies now have led to a capillary setup in which fluorescence is practically eliminated and substantial signal enhancement over standard Raman setups is achieved. With this improved (prototype) setup, detection limits of well below 1 mbar could be obtained in sub-second acquisition times, demonstrating the potential of capillary Raman spectroscopy for real-time, in situ gas sensing and process control applications, down to trace level concentrations. PMID:26378545

  8. Mechanism of ceroid formation in atherosclerotic plaque: in situ studies using a combination of Raman and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Haka, Abigail S.; Kramer, John R.; Dasari, Ramachandra R.; Fitzmaurice, Maryann

    2011-01-01

    Accumulation of the lipid-protein complex ceroid is a characteristic of atherosclerotic plaque. The mechanism of ceroid formation has been extensively studied, because the complex is postulated to contribute to plaque irreversibility. Despite intensive research, ceroid deposits are defined through their fluorescence and histochemical staining properties, while their composition remains unknown. Using Raman and fluorescence spectral microscopy, we examine the composition of ceroid in situ in aorta and coronary artery plaque. The synergy of these two types of spectroscopy allows for identification of ceroid via its fluorescence signature and elucidation of its chemical composition through the acquisition of a Raman spectrum. In accordance with in vitro predictions, low density lipoprotein (LDL) appears within the deposits primarily in its peroxidized form. The main forms of modified LDL detected in both coronary artery and aortic plaques are peroxidation products from the Fenton reaction and myeloperoxidase-hypochlorite pathway. These two peroxidation products occur in similar concentrations within the deposits and represent ~40 and 30% of the total LDL (native and peroxidized) in the aorta and coronary artery deposits, respectively. To our knowledge, this study is the first to successfully employ Raman spectroscopy to unravel a metabolic pathway involved in disease pathogenesis: the formation of ceroid in atherosclerotic plaque.

  9. In-situ tryptophan-like fluorescence: A real-time indicator of faecal contamination in drinking water supplies.

    PubMed

    Sorensen, J P R; Lapworth, D J; Marchant, B P; Nkhuwa, D C W; Pedley, S; Stuart, M E; Bell, R A; Chirwa, M; Kabika, J; Liemisa, M; Chibesa, M

    2015-09-15

    Enteric pathogens are typically inferred from the presence of surrogate indicator organisms such as thermotolerant (faecal) coliforms (TTCs). The analysis of TTCs requires time-consuming incubation in suitable laboratories, which can limit sampling resolution, particularly during critical pollution events. Here, we demonstrate the use of in-situ fluorimeters targeting tryptophan-like compounds as a rapid, reagentless indicator of TTCs in groundwater-derived potable water supplies in Africa. A range of other common indicators of TTCs were also determined including nitrate, turbidity, and sanitary risk survey scores. Sampling was conducted during both the dry and wet seasons to investigate seasonality. Tryptophan-like fluorescence was the most effective predictor of both presence/absence and number of TTCs during both seasons. Seasonal changes in tryptophan-like fluorescence in deeper supplies suggest it is transported more efficiently through the aquifer than TTCs. Moreover, the perennial elevated concentrations in some wells suggest it is more resilient than TTCs in groundwater. Therefore tryptophan-like fluorescence could also be a better indicator of some smaller, more easily transported, and long-lived, pathogenic enteric viruses. These sensors have the potential to be included in real-time pollution alert systems for drinking water supplies throughout the world, as well as for mapping enteric pathogen risks in developing regions. PMID:26026711

  10. In situ forming interpenetrating hydrogels of hyaluronic acid hybridized with iron oxide nanoparticles.

    PubMed

    Kheirabadi, Malihe; Shi, Liyang; Bagheri, Reza; Kabiri, Kourosh; Hilborn, Jöns; Ossipov, Dmitri A

    2015-10-13

    Four derivatives of hyaluronic acid (HA) bearing thiol (HA-SH), hydrazide (HA-hy), 2-dithiopyridyl (HA-SSPy), and aldehyde groups (HA-al) respectively were synthesized. Thiol and 2-dithiopyridyl as well as hydrazide and aldehyde make up two chemically orthogonal pairs of chemo-selective functionalities that allow in situ formation of interpenetrating (IPN) disulfide and hydrazone networks simultaneously upon the mixing of the above derivatives at once. The formation of IPN was demonstrated by comparing it with the formulations of the same total HA concentration but lacking one of the reactive components. The hydrogel composed of all four components was characterized by a larger elastic modulus than those of the control single networks (either disulfide or hydrazone) and the three component formulations gave the softest hydrogels. Moreover, a hydrazone cross-linkage was designed to contain a 1,2-diol fragment. This allowed us to partially disassemble one type of network in the IPN leaving another one unaffected. In particular, treatment of the IPN with either sodium periodate or dithiothreitol resulted in disassembly of the hydrazone and disulfide networks respectively and thus softening of the hydrogel. Contrarily, the single network hydrogels completely dissolved under the corresponding conditions. In corroboration with this, enzymatic degradation of the IPN by hyaluronidase was also substantially slower than the degradation of the single networks. In order to further improve the mechanical properties of the elaborated injectable IPN, it has been in situ hybridized with iron oxide nanoparticles (IONPs). The mesh size of the IPN was smaller than the size of the IONPs resulting in the retention of nanoparticles in the matrix under equilibrium swelling conditions. However, these nanoparticles were released upon enzymatic degradation suggesting their use as MRI tags for non-invasive tracking of the hydrogel material in vivo. Additionally, this injectable hybridized hydrogel with encapsulated IONPs can be used in hyperthermia cancer therapy. PMID:26247066

  11. In-situ Measurements of Colloid Transport and Retention Using Synchroton X-ray Fluorescence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The physics regarding the retention and mobilization of colloids in saturated and unsaturated conditions remains poorly understood, partially due to the inability to measure colloid concentrations in-situ. In this study, we attached Cd+2 ions to clay colloids, and used synchrotron x-rays to cause th...

  12. Fluorescent magnetic hybrid nanoprobe for multimodal bioimaging.

    PubMed

    Koktysh, Dmitry; Bright, Vanessa; Pham, Wellington

    2011-07-01

    A fluorescent magnetic hybrid imaging nanoprobe (HINP) was fabricated by the conjugation of superparamagnetic Fe3O4 nanoparticles and visible light emitting (?600 nm) fluorescent CdTe/CdS quantum dots (QDs). The assembly strategy used the covalent linking of the oxidized dextran shell of magnetic particles to the glutathione ligands of QDs. The synthesized HINP formed stable water-soluble colloidal dispersions. The structure and properties of the particles were characterized by transmission electron and atomic force microscopy, energy dispersive x-ray analysis and inductively coupled plasma optical emission spectroscopy, dynamic light scattering analysis, optical absorption and photoluminescence spectroscopy, and fluorescent imaging. The luminescence imaging region of the nanoprobe was extended to the near-infrared (NIR) (?800 nm) by conjugation of the superparamagnetic nanoparticles with synthesized CdHgTe/CdS QDs. Cadmium, mercury based QDs in HINP can be easily replaced by novel water-soluble glutathione stabilized AgInS2/ZnS QDs to present a new class of cadmium-free multimodal imaging agents. The observed NIR photoluminescence of fluorescent magnetic nanocomposites supports their use for bioimaging. The developed HINP provides dual-imaging channels for simultaneous optical and magnetic resonance imaging. PMID:21597146

  13. Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

    SciTech Connect

    Deutsch, Eric W.; Ball, Cathy; Berman, Jules J.; Bova, G. S.; Brazma, Alvis; Bumgarner, Roger E.; Campbell, David; Causton, Helen C.; Christiansen, Jeff; Daian, Fabrice; Dauga, Delphine; Davidson, Duncan; Gimenez, Gregory; Goo, Young Ah; Grimmond, Sean; Henrich, Thorsten; Herrmann, Bernhard G.; Johnson, Michael H.; Korb, Martin; Mills, Jason C.; Oudes, Asa; Parkinson, Helen E.; Pascal, Laura E.; Pollet, Nicolas; Quackenbush, John; Ramialison, Mirana; Ringwald, Martin; Salgado, David; Sansone, Susanna A.; Sherlock, Gavin; Stoeckert, Christian Jr. J.; Swedlow, Jason; Taylor, Ronald C.; Walashek, Laura; Warford, Anthony; Wilkinson, David G.; Zhou, Yi; Zon, Leonard I.; Liu, Alvin Y.; True, Lawrence D.

    2008-03-03

    We describe the creation process of the Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE). Modeled after the existing minimum information specification for microarray data, we created a new specification for gene expression localization experiments, initially to facilitate data sharing within a consortium. After successful use within the consortium, the specification was circulated to members of the wider biomedical research community for comment and refinement. After a period of acquiring many new suggested requirements, it was necessary to enter a final phase of excluding those requirements that were deemed inappropriate as a minimum requirement for all experiments. The full specification will soon be published as a version 1.0 proposal to the community, upon which a more full discussion must take place so that the final specification may be achieved with the involvement of the whole community.

  14. Nonradioactive in situ hybridization using digoxigenin-labeled oligonucleotides. Applications to musculoskeletal tissues.

    PubMed Central

    Crabb, I. D.; Hughes, S. S.; Hicks, D. G.; Puzas, J. E.; Tsao, G. J.; Rosier, R. N.

    1992-01-01

    We have optimized a technique for in situ localization of specific mRNAs using digoxigenin-11-dUTP-labeled oligonucleotide probes. DNA probes were synthesized for type I and type II collagen as well as transforming growth factor-beta 1 and 2 (TGF beta 1 and TGF beta 2). Control experiments, such as competitive inhibition, nonsense sequence hybridization, and RNAse digestion all indicated that the technique was highly sensitive and specific. In sections of growth plate, type II collagen mRNA was predominantly expressed in the lower proliferative and upper hypertrophic zone, whereas chondrocytes in articular cartilage stained equally. These techniques then were applied to sections cut from archival pathology specimens of musculoskeletal tissues. Primitive chondrocytes in a chondrosarcoma expressed type I and type II collagen mRNA, but did not stain with the nonsense probe. Sections from an osteosarcoma, an aneurysmal bone cyst, and a neurofibroma also were investigated. The ability to use chemically synthesized oligonucleotide probes, the high resolution, and the short development times possible with this in situ procedure makes this technique appealing for applied research into the gene expression of normal and pathologic cellular events. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:1519665

  15. Development of in situ hybridization for the detection of Mycoplasma hyorhinis in formalin-fixed paraffin-embedded tissues from naturally infected pigs with polyserositis.

    PubMed

    Kim, Bongtae; Lee, Kichan; Han, Kiwon; Kim, Duyeol; Ha, Yooncheol; Kim, Chung Hyun; Oh, Yeonsu; Kang, Ikjae; Lee, Jeehoon; Chae, Chanhee

    2010-09-01

    The aim of this study was to develop in situ hybridization for detection of Mycoplasma hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from pigs with polyserositis. M. hyorhinis was isolated from the spleen (2 pigs) and pericardium (1 pig). M. hyorhinis DNA was detected 16 out of 20 pigs with polyserositis. In situ hybridization produced a distinct positive signal for the M. hyorhinis p37 gene in inflammatory cells in the polyserositis. In situ hybridization developed in the present study present diagnostic tools capable of detection of M. hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from the naturally infected pigs. PMID:20424392

  16. In-situ DNA hybridization detection with a reflective microfiber grating biosensor.

    PubMed

    Sun, Dandan; Guo, Tuan; Ran, Yang; Huang, Yunyun; Guan, Bai-Ou

    2014-11-15

    A label-free fiber-optic biosensor with a reflective microfiber Bragg grating (mFBG) configuration for in-situ DNA hybridization detection has been proposed and experimentally demonstrated. A single straight Bragg grating inscribed in the silica microfiber provides two well-defined resonances in reflection, which show different response to external medium refractive index (RI) and present the same temperature sensitivity. By monitoring the wavelength separation between these two resonances, temperature-compensated RI measurement has been achieved. The label-free bio-recognition scheme used demonstrates that the sensor relies on the surface functionalization of a monolayer of poly-l-lysine (PLL), synthetic DNA sequences that bind with high specificity to a given target. In addition to monitoring the surface functionalization of the fiber in real-time, the results also show how the fiber biosensor can detect the presence of the DNA hybridization with high specificity, in various concentration of target DNA solutions, with lowest detectable concentration of 0.5 µM. PMID:24953840

  17. In situ hybridization on whole-mount zebrafish embryos and young larvae.

    PubMed

    Thisse, Bernard; Thisse, Christine

    2014-01-01

    The in situ hybridization uses a labeled complementary RNA strand to localize a specific mRNA sequence in a tissue. This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes. Here we describe a technique that employs in vitro synthesized RNA tagged with digoxigenin uridine-5'-triphosphate (UTP) to determine expression of genes on whole-mount zebrafish embryos and young larvae. Following hybridization, the localization of the specific transcript is visualized immunohistochemically using an anti-digoxigenin antibody conjugated to alkaline phosphatase that hydrolyzes the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 5-bromo-4-chloro-3-indole and inorganic phosphate. 5-Bromo-4-chloro-3-indole can be oxidized by nitro blue tetrazolium (NBT), which forms an insoluble dark blue diformazan precipitate after reduction.This protocol has been used for performing large-scale analyses of the spatial and temporal expression of the zebrafish genome, resulting in the description of more than 8,400 expression patterns that are available at the zebrafish information network (ZFIN.org) in the gene expression section. PMID:25218376

  18. Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation.

    PubMed

    Milián-García, Y; Ramos-Targarona, R; Pérez-Fleitas, E; Sosa-Rodríguez, G; Guerra-Manchena, L; Alonso-Tabet, M; Espinosa-López, G; Russello, M A

    2015-03-01

    Inter-specific hybridization may be especially detrimental when one species is extremely rare and the other is abundant owing to the potential for genetic swamping. The Cuban crocodile (Crocodylus rhombifer) is a critically endangered island endemic largely restricted to Zapata Swamp, where it is sympatric with the widespread American crocodile (C. acutus). An on-island, C. rhombifer captive breeding program is underway with the goals of maintaining taxonomic integrity and providing a source of individuals for reintroduction, but its conservation value is limited by lack of genetic information. Here we collected mtDNA haplotypic and nuclear genotypic data from wild and captive C. rhombifer and C. acutus in Cuba to: (1) investigate the degree of inter-specific hybridization in natural (in situ) and captive (ex situ) populations; (2) quantify the extent, distribution and in situ representation of genetic variation ex situ; and (3) reconstruct founder relatedness to inform management. We found high levels of hybridization in the wild (49.1%) and captivity (16.1%), and additional evidence for a cryptic lineage of C. acutus in the Antilles. We detected marginally higher observed heterozygosity and allelic diversity ex situ relative to the wild population, with captive C. rhombifer exhibiting over twice the frequency of private alleles. Although mean relatedness was high in captivity, we identified 37 genetically important individuals that possessed individual mean kinship (MK) values lower than the population MK. Overall, these results will guide long-term conservation management of Cuban crocodiles for maintaining the genetic integrity and viability of this species of high global conservation value. PMID:25335559

  19. Specific Expression of Aplysia Phosphodiesterase 4 in Bag Cells Revealed by in situ Hybridization Analysis

    PubMed Central

    Jang, Deok-Jin; Kim, Hyoung F.; Sim, Jae-Hoon; Lim, Chae-Seok

    2015-01-01

    Phosphodiesterases (PDEs) play a key role in the regulation of cyclic adenosine monophosphate (cAMP), which in turn mediates various cellular functions including learning and memory. We previously cloned and characterized three PDE4 isoforms (ApPDE4) from Aplysia kurodai. Using reverse transcription polymerase chain reaction (RT-PCR), we found that ApPDE4 isoforms are primarily expressed in the central nervous system. However, the detailed distribution of ApPDE4 mRNA in Aplysia individual ganglions was not evident. In this study, to determine the distribution of ApPDE4 mRNAs in Aplysia ganglions, we performed in situ hybridization (ISH) using a probe targeting ApPDE4, including the PDE catalytic domain. Interestingly, we found the strongest ISH-positive signals in the symmetrical bag cell clusters of the abdominal ganglion. The R2, R14, L7, L2 and L11 neurons in the abdominal ganglion, LP1 neuron in pleural ganglion, and metacerebral (MCC) neurons were ISH-positive. Mechanosensory neurons of the sensory cluster were also stained on the ventral aspect of the right and left pleural ganglia. Taken together, we found the detailed distribution of ApPDE4 mRNA in Aplysia ganglion and support their roles in serotonin (5-HT)-induced synaptic facilitation of Aplysia mechanosensory neurons. PMID:26412974

  20. Rapid in-situ subsurface characterization of a petroleum-contaminated site using laser induced fluorescence and cone penetrometer testing

    SciTech Connect

    Boorse, S.C.

    1996-09-01

    In-situ sampling techniques used to characterize the stratigraphy and extent of subsurface contamination are becoming increasingly common in environmental site investigations. Laser Induced Fluorescence (LIF) combined with Cone Penetrometer Testing (CPT) is a new in-situ technology that provides real-time data on the extent to Aromatic Petroleum Hydrocarbons and stratigraphy in the subsurface. Integrated LIF/CPT data can rapidly provide contaminant and geologic information necessary to define plume boundaries and design efficient and effective remediation plans. The Rapid Optical Screening Tool (ROST{trademark}, a trademark of the Loral Corporation) combines state-of-the-art LIF technology in conjunction with CPT equipment. The combined ROST{trademark}/CPT system provides a two centimeter resolution which quickly and accurately delineates the vertical and horizontal extent of petroleum contamination. The system distinguishes between specific fuel types by obtaining wavelength-time matrices, which are three-dimensional representations of the fluorescence data. By performing the test in-situ, the combined system eliminates cuttings disposal and the cost and time of extensive laboratory analysis associated with other subsurface screening techniques. The ROST{trademark}/CPT system was used at a former refinery to investigate the extent of petroleum hydrocarbons in the subsurface. The ROST{trademark}/CPT tool was hydraulically advanced to an average depth of 30 feet below grade at 150 grid locations in two separate areas. The results provided a clear real-time characterization of the vertical and horizontal extent of three separate plumes and the potential contamination migration pathways. Due to the rapid, continuous subsurface characterization, on-site decision making enabled a 50% reduction in the planned scope of work and significantly reduced the investigation costs to the client.

  1. Imaging of multiple mRNA targets using quantum dot based in situ hybridization and spectral deconvolution in clinical biopsies

    SciTech Connect

    Tholouli, Eleni; Hoyland, Judith A.; Di Vizio, Dolores; O'Connell, Fionnuala; MacDermott, Sarah A.; Twomey, David; Levenson, Richard; Yin, John A. Liu; Golub, Todd R.; Loda, Massimo; Byers, Richard . E-mail: r.byers@manchester.ac.uk

    2006-09-22

    Gene expression mapping using microarray analysis has identified useful gene signatures for predicting outcome. However, little of this has been translated into clinically effective diagnostic tools as microarrays require high quality fresh-frozen tissue samples. We describe a methodology of multiplexed in situ hybridization (ISH) using a novel combination of quantum dot (QD)-labeled oligonucleotide probes and spectral imaging analysis in routinely processed, formalin-fixed paraffin embedded human biopsies. The conditions for QD-ISH were optimized using a poly d(T) oligonucleotide in decalcified bone marrow samples. Single and multiplex QD-ISH was performed in samples with acute leukemia and follicular lymphoma using oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP. Spectral imaging was used for post hybridization tissue analysis, enabling separation of spatially colocalized signals. The method allows quantitative characterization of multiple gene expression using non-bleaching fluorochromes. This is expected to facilitate multiplex in situ transcript detection in routinely processed human clinical tissue.

  2. Branched chain in situ hybridization for albumin as a marker of hepatocellular differentiation: evaluation of manual and automated in situ hybridization platforms

    PubMed Central

    Shahid, Mohammad; Mubeen, Aysha; Tse, Julie; Kakar, Sanjay; Bateman, Adrian; Borger, Darrell; Rivera, Miguel; Ting, David T.; Deshpande, Vikram

    2015-01-01

    Introduction Albumin, widely recognized as a highly sensitive and specific marker of hepatocellular carcinoma (HCC) is currently unavailable in the diagnostic laboratory because of the lack of a robust platform. In a prior study we detected albumin mRNA in the majority of intrahepatic cholangiocarcinomas using a novel branched chain RNA in situ hybridization (ISH) platform. We now explore the utility of albumin ISH as a marker of hepatocellular differentiation in hepatocellular carcinomas, and compare its sensitivity with Hep Par 1 and Arginase-1. Methods We evaluated 93 HCCs and its mimics including neuroendocrine tumors of the gastrointestinal tract (n= 31), neuroendocrine tumors of the pancreas (n= 163), melanoma (n= 15), and gallbladder carcinoma (n=34). We performed ISH for albumin and immunohistochemistry for Hep Par 1 and Arginase-1. Five previously uncharacterized hepatic neoplasms from our files were also evaluated. Immunohistochemistry for Arginase-1 was performed on 59 intrahepatic cholangiocarcinomas. In addition, 43 HCCs evaluated on the manual platform, were also examined on the automated instrument. Results 55% of HCCs were moderately differentiated and 39% poorly differentiated. The sensitivity of ISH for Albumin was 99% with 92 of 93 of HCCs staining positive for albumin. In contrast to ISH, the sensitivity of immunohistochemistry for Hep Par1 and Arginase-1 was 84 % and 83 %, respectively. The sensitivity of albumin for poorly differentiated HCCs was 99%, while that for Arginase-1 and Hep Par 1 was 71% and 64%, respectively. 97% of the HCCs showed albumin positivity in >50% of tumor cells using the ISH platform, as compared to 76% and 70% for Hep Par 1 and Arginase-1 immunohistochemistry, respectively. 3 of the 5 previously uncharacterized neoplasms were positive for albumin ISH. Automated albumin ISH platform performed equivalently to the manual format, with albumin reactivity in >50% of tumor cells in all 43 cases that were tested on both platforms. All non- HCCs were negative for albumin. All 59 intrahepatic cholangiocarcinomas were negative for Arginase-1. Conclusion Branched chain ISH performed on manual and automated mode is a robust assay for detecting albumin with sensitivity for poorly differentiated HCC superior to Arginase-1 and Hep Par 1. When interpreted in conjunction with Arginase-1, albumin ISH offers a high level of sensitivity as well as specificity. PMID:25353287

  3. DNA in situ hybridization for the rapid diagnosis of massive necrotizing avian adenovirus hepatitis and pancreatitis in chicks.

    PubMed

    Goodwin, M A; Latimer, K S; Resurreccion, R S; Miller, P G; Campagnoli, R P

    1996-01-01

    The present study describes the use of DNA in situ hybridization for the rapid diagnosis of massive necrotizing adenovirus hepatitis and pancreatitis in broiler chicks. A light microscope and DNA probes were used to identify avian adenovirus in replicate sections of formalin-fixed paraffin-embedded liver and pancreas from field and experimental chicks. Avian adenovirus infection was confirmed by transmission electron microscopy and virus isolation. PMID:8980813

  4. Cellular localization of simian immunodeficiency virus in lymphoid tissues. II. In situ hybridization.

    PubMed Central

    Wyand, M. S.; Ringler, D. J.; Naidu, Y. M.; Mattmuller, M.; Chalifoux, L. V.; Sehgal, P. K.; Daniel, M. D.; Desrosiers, R. C.; King, N. W.

    1989-01-01

    Lymph nodes and spleens were collected at autopsy and by biopsy from 29 rhesus monkeys infected with simian immunodeficiency virus (SIV). Lymph nodes were classified morphologically into stages of follicular hyperplasia, follicular involution, follicular depletion with normal or expanded paracortices, follicular and paracortical depletion, granulomatous lymphadenitis, or normal. The distribution of SIV RNA was determined by in situ hybridization using a nick translated, 35S labeled, SIVmac DNA probe. Numbers of SIV-infected cells were rare during follicular hyperplasia, numerous during follicular and paracortical expansion, and rare during follicular and paracortical depletion. The splenic morphology reflected that of the lymph nodes; however, the numbers of SIV-positive cells were uniformly lower. SIV RNA was frequently restricted to a single nucleus within multinucleate syncytial cells in two cases of granulomatous lymphadenitis. These results, combined with those of a previous study, provide evidence for antigen trapping in SIV-infected hyperplastic lymph nodes and for widespread viral infection of macrophages and lymphocytes during paracortical expansion. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2537017

  5. A ratiometric fluorescent probe for in situ quantification of basal mitochondrial hypochlorite in cancer cells.

    PubMed

    Hou, Ji-Ting; Li, Kun; Yang, Jin; Yu, Kang-Kang; Liao, Ye-Xin; Ran, Yu-Zhao; Liu, Yan-Hong; Zhou, Xue-Dong; Yu, Xiao-Qi

    2015-04-21

    A ratiometric fluorescent probe () for ClO(-) based on the conjugate of coumarin-rhodamine was presented, which could sense ClO(-) with fast response (within 5 s), high sensitivity and excellent selectivity. More importantly, is the first mitochondria-targeted ratiometric fluorescent probe to image exogenous and endogenous ClO(-). PMID:25785927

  6. Multispectral In-situ Measurements of Organic Matter and Chlorophyll Fluorescence in Seawater: Documenting the Intrusion of the Mississippi River Plume in the West Florida Shelf

    NASA Technical Reports Server (NTRS)

    DelCastillo, Carlos E.; Coble, Paula G.; Conmy, Robyn N.; Mueller-Karger, Frank E.; Vanderbloomen, Lisa; Vargo, Gabriel A.

    2000-01-01

    We performed multispectral in-situ fluorescence measurement of colored dissolved organic matter and chlorophyll in surface water of the West Florida Shelf using West Labs Spectral absorption and Fluorescence Instrument (SAFIre). Continuous measurements underway allowed us to simultaneously map the dispersion of riverine organic material and chlorophyll on the shelf. By using two fluorescence emission ratios we were able to differentiate between riverine and marine CDOM. Our data also showed unusually high concentrations of CDOM offshore. These were attributed to an intrusion of the Mississippi River Plume. We performed limited comparisons between in-situ chlorophyll concentrations measured with SAFIre and chlorophyll values obtained from SeaWiFS satellite data using OC4 and MODIS algorithm. Our results show that, although both algorithms overestimated chlorophyll, MODIS performed better than OC4, particularly in areas with high CDOM concentrations. Analysis of the relationship between chlorophyll and CDOM concentrations within the study area showed regional variability causes by differences in river source.

  7. Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms.

    PubMed

    Randrianjatovo, I; Girbal-Neuhauser, E; Marcato-Romain, C-E

    2015-06-01

    Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 ?g to as high as 50 ?g per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 ?g ml(-1)) showed little or no sugar interference up to 2000 ?g ml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1?±?3.1, 38.3?±?7.1 and 0.3?±?0.1 ?g equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms. PMID:25913004

  8. Detection and localization of syntrophic propionate-oxidizing bacteria in granular sludge by in situ hybridization using 16S rRNA-based oligonucleotide probes.

    PubMed Central

    Harmsen, H J; Kengen, H M; Akkermans, A D; Stams, A J; de Vos, W M

    1996-01-01

    In situ hybridization with fluorescent oligonucleotides was used to detect and localize microorganisms in the granules of two lab-scale upflow anaerobic sludge blanket reactors that had been fed for several months with either sucrose or a mixture of volatile fatty acids. Sections of the granules were hybridized with 16S rRNA-targeted oligonucleotide probes for Bacteria, Archaea, specific phylogenetic groups of methanogens, and two syntrophic propionate-oxidizing strains, MPOB and KOPROP1. Cells of the syntrophic strain KOPROP1 were not detected in either type of sludge granules. Hybridizations of the sucrose-fed granules showed an outer layer of mainly bacterial microcolonies with different morphologies. More inwards of these granules, a layer of different methanogenic microcolonies mixed with large colonies of the syntrophic strain MPOB could be detected. The MPOB colonies were intertwined with hydrogen- or formate-consuming methanogens, indicating their syntrophic growth. The granules fed with volatile fatty acids showed an outer layer of mainly bacteria and then a thick layer of Methanosaeta-like methanogens mixed with a few bacteria and a layer of methanogens mixed with syntrophic MPOB microcolonies. The centers of both sludge types consisted of large cavities and methanogenic microcolonies. These results indicate a juxtapositioning of syntrophic bacteria and methanogens and provide additional evidence for a layered microbial architecture of anaerobic granular sludge. PMID:8633864

  9. In Situ Planetary Mineralogy Using Simultaneous Time Resolved Fluorescence and Raman Spectroscopy

    NASA Technical Reports Server (NTRS)

    Blacksberg, J.; Rossman , G.R.

    2011-01-01

    Micro-Raman spectroscopy is one of the primary methods of mineralogical analysis in the laboratory, and more recently in the field. Because of its versatility and ability to interrogate rocks in their natural form it is one of the front runners for the next generation of in situ instruments designed to explore adverse set of solar system bodies (e.g. Mars, Venus, the Moon, and other primitive bodies such as asteroids and the Martian moons Phobos and Deimos), as well as for pre-selection of rock and soil samples for potential cache and return missions.

  10. Miller-Dieker syndrome resulting from rearrangement of a familial chromosome 17 inversion detected by fluorescence in situ hybridisation.

    PubMed Central

    Kingston, H M; Ledbetter, D H; Tomlin, P I; Gaunt, K L

    1996-01-01

    We report a case of Miller-Dieker syndrome (MDS) owing to an unbalanced rearrangement of a familial pericentric inversion of chromosome 17 (inv(17) (p13.3q25.1)). In addition to lissencephaly and the facial features of MDS, the affected child had other congenital malformations consistent with distal 17q duplication. Initial cytogenetic analysis failed to show any abnormality and fluorescence in situ hybridisation (FISH) studies confirmed the 17p deletion in the proband and identified the chromosome 17 inversion in his mother. FISH studies were performed in other relatives and enabled first trimester prenatal diagnosis by chorionic villus sampling in a subsequent pregnancy of the proband's mother. These findings underline the value of FISH in the investigation of MDS families. Images PMID:8825053

  11. Versatile plug flow catalytic cell for in situ transmission/fluorescence x-ray absorption fine structure measurements

    NASA Astrophysics Data System (ADS)

    Centomo, P.; Meneghini, C.; Zecca, M.

    2013-05-01

    A novel flow-through catalytic cell has been developed for in situ x-ray absorption spectroscopy (XAS) experiments on heterogeneous catalysts under working conditions and in the presence of a liquid and a gas phase. The apparatus allows to carry out XAS measurements in both the transmission and fluorescence modes, at moderate temperature (from RT to 50-80 °C) and low-medium gas pressure (up to 7-8 bars). The materials employed are compatible with several chemicals such as those involved in the direct synthesis of hydrogen peroxide (O2, H2, H2O2, methanol). The versatile design of the cell allows to fit it to different experimental setups in synchrotron radiation beamlines. It was used successfully for the first time to test nanostructured Pd catalysts during the direct synthesis of hydrogen peroxide (H2O2) in methanol solution from dihydrogen and dioxygen.

  12. Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization

    PubMed Central

    Mostegl, Meike M.; Richter, Barbara; Dinhopl, Nora; Weissenböck, Herbert

    2011-01-01

    Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral (Porcine circovirus-2 [PCV-2], Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents (Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable to the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, Cryptosporidium serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared to the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable to 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol. PMID:22362804

  13. Total Water Measurements Using In Situ UV Fragment Fluorescence Spectroscopy in Support of CRYSTAL-FACE

    NASA Technical Reports Server (NTRS)

    Anderson, James G.

    2004-01-01

    Given both the powerful diagnostic importance of the condensed phases of water for dynamics and the impact of phase changes in water on the radiation field, the accurate, in situ observation of total water is of central importance to CRYSTAL-FACE. This is clear both from the defined scientific objectives of the NRA and from developments in the coupled fields of stratosphere/troposphere exchange, cirrus cloud formation/removal and mechanisms for the distribution of water vapor in the middle/upper troposphere. Accordingly, we were funded under NASA Grant NAG5-115487 to perform the following tasks for the CRYSTAL-FACE mission that took place in Key West, Florida, during July 2001: 1) Prepare the Total Water instrument for integration into the WB57F and test flights scheduled for Spring 2002. 2) Calibrate and prepare the Total Water instrument for the Summer 2002 CRYSTAL-FACE science flights based in Jacksonville, Florida. 3) Provide both science and engineering support for the above-mentioned efforts. 4) Analyze and interpret the CRYSTAL-FACE data in collaboration with the other mission scientists. 5) Attend the proposed science workshop in Spring 2003. 6) Publish the data and analysis in peer-reviewed journals.

  14. Fixation/permeabilization procedure for mRNA in situ hybridization of zebrafish whole-mount oocytes, embryos, and larvae.

    PubMed

    Fuentes, Ricardo; Fernández, Juan

    2014-01-01

    A new procedure for improved in situ hybridization of zebrafish whole-mount oocytes, embryos, and early larvae is described. The procedure relies on the simultaneous fixation/permeabilization of samples using formaldehyde as fixative and short C-chain aliphatic carboxylic acids, particularly glacial acetic acid, as permeabilizers. As compared with in situ hybridization performed with routine methods, our procedure is simpler and provides better structural preservation of cells and tissues, equivalent mRNA signals, and similar results in embryos of different developmental stages. It is hypothesized that during aldehyde fixation short C-chain aliphatic carboxylic acids modulate the rate of formation and/or destruction of methylene bridges established between cell proteins. PMID:25218372

  15. An optimized protocol for high-throughput in situ hybridization of zebra finch brain

    PubMed Central

    Carleton, J.; Lovell, P.V.; McHugh, A.; Marzulla, T.; Horback, K.; Mello, C.V.

    2015-01-01

    In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advances in non-radioactive methods based on chromogenic detection of digoxigenin (DIG)-labeled probes have increased spatial resolution compared to emulsion autoradiography, and when paired with high-resolution digital imaging have allowed for the large scale molecular profiling at cellular resolution within a histological context (e.g. Allen Brain Atlas, GenePaint.org; (Visel et al. 2004; Lein et al. 2007). However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes a low cost, small footprint, high-throughput ISH procedure for 10 ?m sections developed to document brain gene expression in zebra finches (http://www.zebrafinchatlas.org). It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium (Replogle et al. 2008) and is based on previously described protocols for radiolabeled riboprobes (Clayton et al. 1988; Mello and Clayton 1994; Mello et al. 1997) that we have adapted for DIG-labeled (Lovell and Mello 2011; Lovell et al. 2013). Conditions have now been further optimized to produce cellular labeling approaching the resolution of immunohistochemical methods, low background, and compatibility with high-resolution digital imaging. This protocol allows a technician to process ~180 slides per week, and can be scaled to accommodate a broad range of tissues for which cryosections can be obtained. PMID:25342071

  16. [Chromosomal structure of the hybrids between Allium cepa L. and Allium fistulosum L. with relative resistance to downy mildew based on in situ hybridization].

    PubMed

    Budylin, M V; Kan, L Iu; Romanov, V S; Khrustaleva, L I

    2014-04-01

    Genomic in situ hybridization (GISH) was used for a chromosomal composition study of the later generations of interspecific hybrids between A. cepa L. and A. fistulosum L., which are relatively resistant to downy mildew (peronosporosis). GISH revealed that F2 hybrids, which did not produce seeds, were triploids (2n = 3x = 24) with 24 chromosomes and possessed in their compliments 16 chromosomes of A. fistulosum L. and eight chromosomes of A. cepa L. or eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L. The advanced F5 hybrid, which produced few seeds, was amphidiploid with 32 chromosomes. BC1F5 hybrid was triploid with eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L., which did not produce seeds. BC2 (BC1F5) plant was amphidiploid that possessed 4 recombinant chromosomes and produced few seeds. GISH results point to 2n-gametes formation in macro- and microsporogenesis of the hybrids. The mechanism of 2n-gametes formation and the possibility of apomixes events in the backcrossing progeny are discussed. PMID:25715446

  17. A study of myc-related gene expression in small cell lung cancer by in situ hybridization.

    PubMed Central

    Gu, J.; Linnoila, R. I.; Seibel, N. L.; Gazdar, A. F.; Minna, J. D.; Brooks, B. J.; Hollis, G. F.; Kirsch, I. R.

    1988-01-01

    The expression of myc-related genes (c-myc, N-myc, and L-myc) in small cell lung cancer (SCLC) was studied by RNA-RNA tissue in situ hybridization. The tissues investigated included cytospins of ten cell lines derived from patients with SCLC, four corresponding nude mouse xenografts from cell lines, and metastatic tumor tissue obtained by surgical biopsy and at autopsy. The probes were prepared as 35S labeled complementary RNA. The expression of each gene was demonstrated specifically by autoradiography in the cytoplasm of the neoplastic cell samples. The average levels of oncogene expression in each specimen corroborated previous data obtained by Northern blot assays. In addition, heterogeneity in gene expression from cell to cell in each sample was noted. This study represents the first attempt to demonstrate oncogene expression in lung cancer cell lines and tissues in situ, and confirms that the expression of these myc related genes can be seen in the primary tumor. The technique of RNA-RNA tissue in situ hybridization has great potential in answering fundamental questions of tumor cell heterogeneity and progression in SCLC. It should be useful in both prospective and retrospective studies. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2456019

  18. In-situ speciation of arsenic contaminated soil using micro-focused x-ray fluorescence and x-ray absorption fine

    E-print Network

    Sparks, Donald L.

    In-situ speciation of arsenic contaminated soil using micro-focused x-ray fluorescence and x-A, 0-20 cm; LM-B, 20-40 cm) of a mixed metal-arsenic contaminated soil from a former copper chromated-contaminating metal cations (Cu, Zn, & Cr) in the solid phase speciation of arsenic. Elemental maps from ÝSXRF

  19. The effects of particles and dissolved materials on in situ algal pigment fluorescence sensors

    NASA Astrophysics Data System (ADS)

    Saraceno, J.; Bergamaschi, B. A.; Downing, B. D.

    2013-12-01

    Field deployable sensors that measure algal pigment fluorescence (APF), such as chlorophyll-a (excitation/emission ca. 470/685 nm), and phycocyanin (ca. 590/685 nm), have been used to estimate algal biomass and study food-web dynamics in coastal and oceanic waters for many years. There is also widespread use of these sensors in real time river-observing networks. However, freshwater systems often possess elevated levels of suspended solids and dissolved organic material that can interfere with optical measurements. Data collected under conditions that result in interferences may not be comparable across time and between sites unless the data are appropriately corrected. Using standard reference materials and a surrogate for algal fluorescence (Rhodamine WT), lab experiments were conducted on several commercially available sensors to quantify sensitivity to interferences over a range of naturally occurring surface water conditions (DOC : 0-30 mg/L and turbidity: 0- 1000 FNU ). Chlorophyll-a sensors exhibited a slight but significant positive bias (<1%) at DOC concentrations < 2 mg/L, and a negative, non-linear bias at DOC concentrations >2 mg/L, with signal quenching reaching a maximum of 15% at 30 mg/L DOC. All phycocyanin sensors displayed a positive non-linear bias with DOC concentration, reaching a maximum of 40% difference at 30 mg/L DOC. Both chlorophyll-a and phycocyanin sensors showed a positive linear relationship with suspended solids concentration (as indicated by turbidity).The effect of suspended solids on APF output can be explained by the detection of scattered excitation light (leaking through emission filters). Similar qualitative effects were observed for the sensors tested, though the magnitude of the effect varied among sensor type. This indicates that differences in sensor designs such as geometry, wavelength and signal post processing techniques is related to its sensitivity to interferences. Although sensors exhibited significant cross sensitivity to interferences, our results indicate that simple corrections can largely remove sensor bias. To remove bias due to optical interferences and generate high quality, repeatable APF data, knowledge of the optical properties of the matrix and/or coincident measures of the concentration of suspended solids and dissolved organics (through surrogates such as turbidity and colored dissolved organic matter (cDOM) fluorescence, respectively), are typically needed.

  20. Scalable In Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers

    PubMed Central

    Kiflemariam, Sara; Andersson, Sandra; Asplund, Anna; Pontén, Fredrik; Sjöblom, Tobias

    2012-01-01

    Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues. PMID:22412953

  1. Far-field disentanglement of modes in hybrid plasmonic-photonic crystals by fluorescence nano-reporters

    NASA Astrophysics Data System (ADS)

    Ungureanu, Simona; Kolaric, Branko; Chen, Jianing; Hillenbrand, Rainer; Vallée, Renaud A. L.

    2013-07-01

    In this paper, the resonance modes exhibited by a hybrid nanostructure have been disentangled in the far-field owing to narrow-band fluorescence nano-reporters. Hybrid plasmonic-photonic crystals were fabricated using large (457 nm) monodisperse polystyrene spheres self-assembled into 2D photonic crystals and subsequently coated by a 30 nm thick silver layer. Such structures exhibit a complex resonance pattern, which has been elucidated owing to numerical simulations and electric near-field patterns obtained with a scattering type scanning near-field optical microscope (s-SNOM). For the sake of disentangling the resonance modes of the hybrid structure in the far-field, different types of semiconductor quantum dots (QDs), acting as nano-reporters of the local interactions, were dispersed on top of distinct structures. Depending on the relative overlap of the emission spectrum of a particular type of QDs with the resonance features of the hybrid structure, we affect their emission rate in a unique way, as a consequence of the complex interaction occurring between the plasmo-photonic modes and the excitons. Such plasmonic structures appear to be particularly relevant for fluorescence-based sensing devices.

  2. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, Robert B. (Salt Lake City, UT); Kimball, Alvin W. (Salt Lake City, UT); Gesteland, Raymond F. (Salt Lake City, UT); Ferguson, F. Mark (Salt Lake City, UT); Dunn, Diane M. (West Valley City, UT); Di Sera, Leonard J. (Salt Lake City, UT); Cherry, Joshua L. (Salt Lake City, UT)

    1995-01-01

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

  3. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

    1995-11-28

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

  4. Synchrotron micro-X-ray fluorescence analysis of natural diamonds: First steps in identification of mineral inclusions in situ

    SciTech Connect

    Sitepu, Husin; Kopylova, Maya G.; Quirt, David H.; Cutler, Jeffrey N.; Kotzer, Thomas G.

    2008-06-09

    Diamond inclusions are of particular research interest in mantle petrology and diamond exploration as they provide direct information about the chemical composition of upper and lower mantle and about the petrogenetic sources of diamonds in a given deposit. The objective of the present work is to develop semi-quantitative analytical tools for non-destructive in situ identification and characterization of mineral inclusions in diamonds using synchrotron micro-X-ray Fluorescence ({mu}SXRF) spectroscopy and micro-X-ray Absorption Near Edge Structure ({mu}XANES) spectroscopy at a focused spot size of 4 to 5 micrometers. The data were collected at the Pacific Northwest Consortium (PNC-CAT) 20-ID microprobe beamline at the Advanced Photon Source, located at the Argonne National Laboratory, and yielded the first high-resolution maps of Ti, Cr, Fe, Ni, Cu, and Zn for natural diamond grains, along with quantitative {mu}SXRF analysis of select chemical elements in exposed kimberlite indicator mineral grains. The distribution of diamond inclusions inside the natural diamond host, both visible and invisible using optical transmitted-light microscopy, can be mapped using synchrotron {mu}XRF analysis. Overall, the relative abundances of chemical elements determined by {mu}SXRF elemental analyses are broadly similar to their expected ratios in the mineral and therefore can be used to identify inclusions in diamonds in situ. Synchrotron {mu}XRF quantitative analysis provides accurate estimates of Cr contents of exposed polished minerals when calibrated using the concentration of Fe as a standard. Corresponding Cr K-edge {mu}XANES analyses on selected inclusions yield unique information regarding the formal oxidation state and local coordination of Cr.

  5. Electron microscopic in situ hybridization and autoradiography: Localization and transcription of rDNA in human lymphocyte nucleoli

    SciTech Connect

    Wachtler, F.; Mosgoeller, W.S.; Schwarzacher, H.G. )

    1990-04-01

    The distribution of ribosomal DNA (rDNA) in the nucleoli of human lymphocytes was revealed by in situ hybridization with a nonautoradiographic procedure at the electron microscopic level. rDNA is located in the dense fibrillar component of the nucleolus but not in the fibrillar centers. In the same cells the incorporation of tritiated uridine takes place in the dense fibrillar component of the nucleolus as seen by autoradiography followed by gold latensification. From these findings it can be concluded that the transcription of ribosomal DNA takes place in the dense fibrillar component of the nucleolus.

  6. Sex chromosome loss and aging: in situ hybridization studies on human interphase nuclei.

    PubMed Central

    Guttenbach, M; Koschorz, B; Bernthaler, U; Grimm, T; Schmid, M

    1995-01-01

    A total of 1,000 lymphocyte interphase nuclei per proband from 90 females and 138 males age 1 wk to 93 years were analyzed by in situ hybridization for loss of the X and Y chromosomes, respectively. Both sex chromosomes showed an age-dependent loss. In males, Y hypoploidy was very low up to age 15 years (0.05%) but continuously increased to a frequency of 1.34% in men age 76-80 years. In females, the baseline level for X chromosome loss is much higher than that seen for the Y chromosome in males. Even prepubertal females show a rate of X chromosome loss, on the order of 1.5%-2.5%, rising to approximately 4.5%-5% in women older than 75 years. Dividing the female probands into three biological age groups on the basis of sex hormone function (< 13 years, 13-51 years, and > 51 years), a significant correlation of X chromosome loss versus age could clearly be demonstrated in women beyond age 51 years. Females age 51-91 years showed monosomy X at a rate from 3.2% to 5.1%. In contrast to sex chromosomal loss, the frequency of autosomal monosomies does not change during the course of aging: Chromosome 1 and chromosome 17 monosomic cells were found with a constant incidence of 1.2% and 1%, respectively. These data also indicate that autosome loss in interphase nuclei is not a function of chromosome size. Images Figure 1 Figure 2 PMID:7485166

  7. Sex chromosome loss and aging: In situ hybridization studies on human interphase nuclei

    SciTech Connect

    Guttenbach, M.; Koschorz, B.; Bernthaler, U.

    1995-11-01

    A total of 1,000 lymphocyte interphase nuclei per proband from 90 females and 138 males age 1 wk to 93 years were analyzed by in situ hybridization for loss of the X and Y chromosomes, respectively. Both sex chromosomes showed an age-dependent loss. In males, Y hypoploidy was very low up to age 15 years (0.05%) but continuously increased to a frequency of 1.34% in men age 76-80 years. In females, the baseline level for X chromosome loss is much higher than that seen for the Y chromosome in males. Even prepubertal females show a rate of X chromosome loss on the order of 1.5%-2.5%, rising to {approximately}4.5%-5% in women older than 75 years. Dividing the female probands into three biological age groups on the basis of sex hormone function (<13 years, 13-51 years, and >51 years), a significant correlation of X chromosome loss versus age could clearly be demonstrated in women beyond age 51 years. Females age 51-91 years showed monosomy X at a rate from 3.2% to 5.1%. In contrast to sex chromosomal loss, the frequency of autosomal monosomies does not change during the course of aging: chromosome 1 and chromosome 17 monosomic cells were found with a constant incidence of 1.2% and 1%, respectively. These data also indicate that autosome loss in interphase nuclei is not a function of chromosome size. 34 refs., 5 figs., 6 tabs.

  8. Expression of mast-cell-specific proteases in tissues of mice studied by in situ hybridization.

    PubMed Central

    Jippo, T.; Tsujino, K.; Kim, H. M.; Kim, D. K.; Lee, Y. M.; Nawa, Y.; Kitamura, Y.

    1997-01-01

    The protease mRNA expression phenotype of individual mast cells was studied by in situ hybridization. Mouse mast cell protease (MMCP)-2 mRNA was expressed by mast cells located in the mucosa of the stomach of WB(-)+/+ and (WB x C57BL/6)F1(-)+/+ (hereafter WBB6F1(-)+/+) mice but not by mast cells in the same tissue of C57BL/ 6(-)+/+ mice. Even in the stomach of WBB6F1(-)+/+ mice, mast cells located in the muscularis propria did not express MMCP-2 mRNA. The mRNAs of MMCP-4 and mouse mast cell carboxypeptidase A were not expressed by mast cells in the stomach mucosa of untreated WBB6F1(-)+/+ mice but were expressed after the infection of Strongyloides venezuelensis. We examined whether MMCP-2 mRNA expression varied by changing environments of mast cells. Cultured mast cells of WBB6F1(-)+/+ mice that expressed MMCP-2 mRNA were transplanted into the stomach wall of genetically mast-cell-deficient WBB6F1(-)W/Wv mice. Mast cells that appeared in the mucosa expressed the MMCP-2 mRNA, but mast cells that appeared in the muscularis propria did not, indicating the adaptation of cultured mast cells into a new environment. In contrast to cultured mast cells, peritoneal mast cells of WBB6F1(-)+/+ mice that expressed MMCP-2 mRNA as well did not adapt to the muscularis propria of WBB6F(1)-W/Wv mice. The MMCP-2 mRNA remained to be expressed after the settlement in either the mucosa or the muscularis propria. Furthermore, the peritoneal mast cells did not change the MMCP-4 and MMCP-6 mRNA expression phenotype after the settlement in either the mucosa or the muscularis propria of WBB6F(1)-W/Wv mice. The present result indicated that both intracellular factors such as strain specificity and source of mast cells and extracellular factors such as tissue specificity and helminth infection influenced the protease expression phenotypes. Images Figure 1 Figure 2 Figure 3 PMID:9094993

  9. Hybridization may facilitate in situ survival of endemic species through periods of climate change

    NASA Astrophysics Data System (ADS)

    Becker, Matthias; Gruenheit, Nicole; Steel, Mike; Voelckel, Claudia; Deusch, Oliver; Heenan, Peter B.; McLenachan, Patricia A.; Kardailsky, Olga; Leigh, Jessica W.; Lockhart, Peter J.

    2013-12-01

    Predicting survival and extinction scenarios for climate change requires an understanding of the present day ecological characteristics of species and future available habitats, but also the adaptive potential of species to cope with environmental change. Hybridization is one mechanism that could facilitate this. Here we report statistical evidence that the transfer of genetic information through hybridization is a feature of species from the plant genus Pachycladon that survived the Last Glacial Maximum in geographically separated alpine refugia in New Zealand's South Island. We show that transferred glucosinolate hydrolysis genes also exhibit evidence of intra-locus recombination. Such gene exchange and recombination has the potential to alter the chemical defence in the offspring of hybridizing species. We use a mathematical model to show that when hybridization increases the adaptive potential of species, future biodiversity will be best protected by preserving closely related species that hybridize rather than by conserving distantly related species that are genetically isolated.

  10. Development of the Minimum Information Specification for in situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

    SciTech Connect

    Deutsch, Eric W.; Ball, Catherine A.; Bova, G. Steven; Brazma, Alvis; Bumgarner, Roger E.; Campbell, David; Causton, Helen C.; Christiansen, Jeff; Davidson, Duncan; Eichner, Lillian J.; Goo, Young Ah; Grimmond, Sean; Henrich, Thorsten; Johnson, Michael H.; Korb, Martin; Mills, Jason C.; Oudes, Asa; Parkinson, Helen E.; Pascal, Laura E.; Quackenbush, John; Ramialison, Mirana; Ringwald, Martin; Sansone, Susanna A.; Sherlock, Gavin; Stoeckert, Christian Jr. J.; Swedlow, Jason; Taylor, Ronald C.; Walashek, Laura; Zhou, Yi; Liu, Alvin Y.; True, Lawrence D.

    2006-06-06

    Background One purpose of the biomedical literature is to report results in sufficient detail so that the methods of data collection and analysis can be independently replicated and verified. In order to ensure that this level of detail is provided in published works, a minimum information specification is needed for each experimental data type and for this specification to be a requirement for publication in peer-reviewed journals. This is especially beneficial for researchers working with complex data types and experiments. A data content specification has already been widely accepted by, and directly benefited, the microarray community, and efforts are well underway to develop a comparable specification for proteomics data types. However, no similar specification exists for visual interpretation-based tissue protein and transcript abundance/localization experiments (hereafter referred to as ‘gene expression localization experiments’), such as in situ hybridization and experiments involving immunohistochemistry. Results Here we present for consideration a specification, called the “Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)”. It is modelled after the MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments. Data specifications like MIAME and MISFISHIE specify the information content without specifying a format for encoding that information. The MISFISHIE specification describes six types of information that should be provided for each gene expression localization experiment: Experimental Design, Biomaterials and Treatments, Reporters, Staining, Imaging Data, and Image Characterizations. A general checklist is provided for quick and easy reference and to promote adherence to the specification. We consider that most articles describing gene expression localization studies do not fully provide the minimum information needed for independent verification of results. In a small survey of 32 journal articles from the past five years, we found that approximately 90% of them did not meet all the requirements, although many met most of them. We propose that requiring authors to provide the minimum experimental detail about gene expression localization studies would facilitate reproducibility and interpretability of results by fellow investigators. Furthermore, inclusion of specific experimental details such as reagents and methods in publications would ultimately allow others to readily search the literature for these data items, especially given the ongoing trend towards open access full text journals. Conclusion This specification was initially developed by members of the NIH/NIDDK Stem Cell Genome Anatomy Projects consortium (http://www.scgap.org ) to facilitate data sharing within the consortium. Use of the specification has benefited the consortium and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal. Should the community accept the final proposal, we would encourage reviewers, journal editors and funding agencies to promote compliance with MISFISHIE for all studies that report gene expression localization data so that all published data and resulting conclusions may be correctly interpreted, and that independent investigators would have the necessary information that would enable them to repeat the experiment. More information and examples may be obtained at http://mged.sourceforge.net/misfishie/.

  11. Fluorescence in situ hybridisation analysis of bone marrow trephine biopsy specimens; an additional tool in the diagnostic armoury.

    PubMed

    Neat, Michael J; Moonim, Mufaddal T; Dunn, Robert G; Geoghegan, Helen; Foot, Nicola J

    2013-01-01

    Fluorescence in situ hybridisation (FISH) analysis is now widely employed in the diagnosis and risk stratification of a wide range of malignant diseases. While this technique is used successfully with formalin-fixed paraffin-embedded (FFPE) sections from numerous tissue types, FISH analysis of FFPE tissue sections from trephine biopsy specimens has been less widely reported, possibly due to technical limitations relating to the decalcification protocols employed. During the last 4 years FISH analysis has been carried out successfully in 42 out of 55 (76%) consecutive trephine biopsy specimens received as part of the standard diagnostic service at our institution. Samples decalcified using EDTA-based protocols were analysed successfully in 31/31 cases (100%), whereas only 11/24 samples (46%) decalcified using formic acid-based protocols were successful. In our experience, FISH analysis of trephine biopsy specimens is a highly reproducible technique and a very useful adjunctive tool in the diagnostic armoury; however, its use in a standard diagnostic setting relies on the use of EDTA-based decalcification protocols. PMID:23038690

  12. In situ hybridization of nucleus basalis neurons shows increased. beta. -amyloid mRNA in Alzheimer disease

    SciTech Connect

    Cohen, M.L.; Golde, T.E.; Usiak, M.F.; Younkin, L.H.; Younkin, S.G.

    1988-02-01

    To determine which cells within the brain produce ..beta..-amyloid mRNA and to assess expression of the ..beta..-amyloid gene in Alzheimer disease, the authors analyzed brain tissue from Alzheimer and control patients by in situ hybridization. The results demonstrate that ..beta..-amyloid mRNA is produced by neurons in the nucleus basalis of Meynert and cerebral cortex and that nuclues basalis perikarya from Alzheimer patients consistently hybridize more ..beta..-amyloid probe than those from controls. These observations support the hypothesis that increased expression of the ..beta..-amyloid gene plays an important role in the deposition of amyloid in the brains of patients with Alzheimer disease.

  13. FISHing Tips: What Every Clinician Should Know About 1p19q Analysis in Gliomas Using Fluorescence in situ Hybridisation.

    PubMed

    Pinkham, M B; Telford, N; Whitfield, G A; Colaco, R J; O'Neill, F; McBain, C A

    2015-08-01

    1p19q co-deletion is a chromosomal alteration associated with primary brain tumours of oligodendroglial histology. It is an established predictive and prognostic biomarker that informs whether patients are offered radiotherapy, chemotherapy or both. In the near future, 1p19q co-deletion status may also be incorporated into the reclassification of gliomas. Analysis is commonly carried out using fluorescence in situ hybridisation (FISH) because it is a reliable and validated laboratory technique. The result is generally considered to be dichotomous (1p19q co-deletion present or absent), but there are subtleties in interpretation that are of clinical relevance. Separate centres may interpret certain chromosome deletion patterns differently. Pivotal trials in mixed and pure anaplastic oligodendrogliomas have used slightly different FISH probe ratios as the cut-off for chromosome deletion. Here we review the clinical implications of this variability and review the process of 1p19q co-deletion assessment using FISH in gliomas from a clinician's perspective. We also consider common alternative methods of analysis. PMID:25971646

  14. FMT-XCT: in vivo animal studies with hybrid fluorescence molecular tomography-X-ray computed tomography.

    PubMed

    Ale, Angelique; Ermolayev, Vladimir; Herzog, Eva; Cohrs, Christian; de Angelis, Martin Hrabé; Ntziachristos, Vasilis

    2012-06-01

    The development of hybrid optical tomography methods to improve imaging performance has been suggested over a decade ago and has been experimentally demonstrated in animals and humans. Here we examined in vivo performance of a camera-based hybrid fluorescence molecular tomography (FMT) system for 360° imaging combined with X-ray computed tomography (XCT). Offering an accurately co-registered, information-rich hybrid data set, FMT-XCT has new imaging possibilities compared to stand-alone FMT and XCT. We applied FMT-XCT to a subcutaneous 4T1 tumor mouse model, an Aga2 osteogenesis imperfecta model and a Kras lung cancer mouse model, using XCT information during FMT inversion. We validated in vivo imaging results against post-mortem planar fluorescence images of cryoslices and histology data. Besides offering concurrent anatomical and functional information, FMT-XCT resulted in the most accurate FMT performance to date. These findings indicate that addition of FMT optics into the XCT gantry may be a potent upgrade for small-animal XCT systems. PMID:22561987

  15. Developments in laser-induced fluorescence spectroscopy for quantitative in situ measurements of free radicals in the troposphere

    NASA Astrophysics Data System (ADS)

    Heard, Dwayne

    2015-04-01

    Photo-oxidation in the troposphere is highly complex, being initiated by short lived free radical species, in the daytime dominated by the hydroxyl radical, OH. Chemical oxidation cycles, which also involve peroxy radicals (HO2 and RO2), remove natural or anthropogenic emissions (for example methane) and generate a range of secondary products, for example ozone, nitrogen dioxide, acidic and multifunctional organic species, and secondary organic aerosol, which impact on human health and climate. Owing to their short lifetime in the atmosphere, the abundance of radicals is determined solely by their rate of chemical production and loss, and not by transport. Field measurements of the concentrations of radicals and comparison with calculations using a numerical model therefore constitutes one of the very best ways to test whether the chemistry in each of these locations is understood and accurately represented in the model. Validation of the chemistry is important, as the predictions of climate and air quality models containing this chemistry are used to drive the formulation of policy and legislation. However, in situ measurements of radical species, owing to their very low abundance (often sub part per trillion) and short lifetimes (< 1 second for OH), remain extremely challenging. Laser-induced fluorescence spectroscopy (LIF) has enjoyed considerable success worldwide for the quantitative detection of radicals in a range of environments. The radicals are either excited directly by the laser (e.g. OH, IO) or are first chemically converted to OH prior to detection (e.g. HO2, RO2). Recent developments in the LIF technique for radical detection, which uses a supersonic expansion with detection at low pressure and multi kHz pulse repetition rate tunable laser systems, will be discussed, together with calibration methods to make signals absolute, and identification of potential interferences. LIF instruments have been operated on ground, ship and aircraft platforms at a number of locations worldwide, and examples from recent fieldwork involving the Leeds instruments will be presented.

  16. Characterization of the cellular origin of a tissue-engineered human phalanx model by in situ hybridization.

    PubMed

    Chubinskaya, Susan; Jacquet, Robin; Isogai, Noritaka; Asamura, Shinichi; Landis, William J

    2004-01-01

    Tissue-engineered models of human phalanges have previously been fabricated from a combination of bovine periosteum, cartilage, tendon, and biodegradable polyglycolic acid and poly-L-lactic acid scaffolds. Resulting constructs implanted in athymic mice for more than 40 weeks developed new bone, cartilage, and tendon and became vascularized, but cell types comprising the constructs were unidentified. The origin of cells in middle phalanx models implanted for 20 weeks in nude mice has been studied by in situ hybridization analyzing species-specific gene expression. Oligonucleotide probes homologous to species-specific gene sequences of bovine type II and X collagen, aggrecan, bone sialoprotein, biglycan, and osteopontin, and mouse decorin were labeled with (35)S and hybridized to respective serial sections of bovine tissue, mouse tissue, and phalanx constructs. In situ hybridization showed positive message and tissue-specific localization for all bovine-specific probes examined within cartilaginous and midshaft portions of constructs and negative message for the mouse-specific decorin probe. These data show that osteoblasts and chondrocytes comprising constructs are derived exclusively from their original bovine sources over 20 weeks of implantation. Defining the cellular origin of the models lends insight into their biological, chemical, and physical nature and their growth and development. Maintenance of their initial genotype is crucial for future application of the models in augmenting impaired human phalanges and related tissues. PMID:15363176

  17. pH-Sensitive polymer assisted self-aggregation of bis(pyrene) in living cells in situ with turn-on fluorescence

    NASA Astrophysics Data System (ADS)

    Duan, Zhongyu; Gao, Yu-Juan; Qiao, Zeng-Ying; Qiao, Shenglin; Wang, Yongmei; Hou, Chunyuan; Wang, Lei; Wang, Hao

    2015-09-01

    Supramolecular self-assemblies with various nanostructures in organic and aqueous solutions have been prepared with desired functions. However, in situ construction of self-assembled superstructures in physiological conditions to achieve expected biological functions remains a challenge. Here, we report a supramolecular system to realize the in situ formation of nanoaggregates in living cells. The bis(pyrene) monomers were dispersed inside of hydrophobic domains of pH-sensitive polymeric micelles and delivered to the lysosomes of cells. In the acidic lysosomes, the bis(pyrene) monomers were released and self-aggregated with turn-on fluorescence. We envision this strategy for in situ construction of supramolecular nanostructures in living cells will pave the way for molecular diagnostics in the future.

  18. High-Sensitivity In situ Fluorescence Imaging of Ytterbium Atoms in a Two-Dimensional Optical Lattice with Dual Optical Molasses

    NASA Astrophysics Data System (ADS)

    Shibata, Kosuke; Yamamoto, Ryuta; Takahashi, Yoshiro

    2014-01-01

    We developed a dual molasses technique which enabled us to perform high-sensitivity in situ fluorescence imaging of ytterbium (Yb) atoms in a two-dimensional optical lattice prepared in a thin glass cell. This technique successfully combines two different kinds of optical molasses for Yb atoms, that is, the one using the 1S0-1P1 transition which provides high-resolution in the in situ fluorescence imaging and the other using the 1S0-3P1 transition for cooling the atoms in the optical lattice. We performed in situ imaging of 174Yb atoms and could observe a Moiré pattern with a period of about 6 µm produced by the molasses beam with 556 nm and the optical lattice with 532 nm, which implies that the temperature was kept below the lattice depth during the fluorescence imaging. The number of photons per atom is estimated to be enough for single atom detection with our imaging system. This result is quite promising for the realization of an Yb quantum gas microscope.

  19. Detection of HTLV-1 by polymerase chain reaction in situ hybridization in adult T-cell leukemia/lymphoma.

    PubMed Central

    Setoyama, M.; Kerdel, F. A.; Elgart, G.; Kanzaki, T.; Byrnes, J. J.

    1998-01-01

    A method for nonradioactive polymerase chain reaction in situ hybridization was developed and used to determine the distribution of human T-lymphotropic virus type I (HTLV-I) proviral DNA in paraffin-embedded surgical specimens of adult T-cell leukemia/lymphoma (ATLL). As controls, we used biopsy samples of five cases of mycosis fungoides, cells of an HTLV-I-infected cell line (MT2), as well as HTLV-1-negative cells (YAS). We successfully detected the amplicon of the HTLV-1 tax sequence in the nuclei of the cutaneous infiltrating lymphoid cells in 90% (9/10) of ATLL cases. Studies also revealed the existence of HTLV-1 provirus DNA in nuclei of sweat gland epithelial cells and vascular endothelial cells as well as lymphoid cells in ATLL patients. Mycosis fungoides and YAS cells were negative for the HTLV-I tax sequence, but MT2 cells were strongly positive. The results indicated that this technique was more sensitive in detecting intracellular amplicons than was the previous in situ hybridization method. Through its use, we were able to easily determine the distribution of HTLV-I-positive cells among the various cells and tissues of paraffin-embedded archival materials. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9502410

  20. Compact hybrid (gold nanodendrite-quantum dots) assembly: plasmon enhanced fluorescence-based platform for small molecule sensing in solution.

    PubMed

    Chen, Huide; Xia, Yunsheng

    2014-11-18

    In this study, we have presented a novel plasmon enhanced fluorescence (PEF) system for label-free sensing of small molecules in bulk solution. The amine-terminated gold nanodendrite (AuND) and carboxyl-terminated QDs directly assemble each other by amine-carboxyl attraction. Without any spacer layers, PEF can be increased by 4 times during the formation of the compact hybrid (AuND-QDs) assembly. Both experiment and finite-difference time domain calculation results indicate that the distinct solution-PEF effect is ascribed to two reasons: (1) The used AuNDs simultaneously possess four features in morphology and topology, well-defined superstructure, sharp tips and edges, moderately elongated subunits, and smaller size. (2) The hybrid (AuND-QDs) assembly has a very compact structure. So, the fluorescence is well enhanced by the effective increase of excitation and radiative decay rates with the decrease of scattering effect. The (AuND-QDs) assembly is then employed for sensing of trinitrotoluene (TNT), one of the highly explosive and environmentally detrimental substances, in bulk solution. The sensing principle is that the analytes can react with primary amines on the AuND surface and form Meisenheimer complexes, which break the preformed assemblies and result in the fluorescence recovery of the QDs. The linear range is 0-8.8 nM with 0.05 nM detection limit. The present quasi-picomole level sensitivity is one of the best results for fluorescent TNT sensing. The developed method is successfully applied to TNT sensing in real environmental samples, indicating the practical potential. PMID:25317671

  1. Normal development of the tomato clownfish Amphiprion frenatus: live imaging and in situ hybridization analyses of mesodermal and neurectodermal development.

    PubMed

    Ghosh, J; Wilson, R W; Kudoh, T

    2009-12-01

    The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal development. Both morphology of live embryos and tissue-specific staining revealed significant differences in the gross developmental programme of A. frenatus compared with better-known teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation of melanophores at the 10-15 somite stage and a distinctive pattern of melanophore distribution. These results suggest evolutionary adaptability of the teleost developmental programme. The ease of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported characteristics make A. frenatus a potentially important model marine fish species for studying embryonic development, physiology, ecology and evolution. PMID:20738687

  2. Real-Time Observation of Platinum Redispersion on Ceria-Based Oxide by In-situ Turbo-XAS in Fluorescence Mode

    SciTech Connect

    Nagai, Yasutaka; Dohmae, Kazuhiko; Tanabe, Toshitaka; Shinjoh, Hirofumi; Takagi, Nobuyuki; Ikeda, Yasuo; Guilera, Gemma; Pascarelli, Sakura; Newton, Mark; Matsumoto, Shin'ichi

    2007-02-02

    A real-time observation of the redispersion behavior of sintered Pt on ceria-based oxide was made possible by in-situ time-resolved Turbo-XAS in fluorescence mode. 2 wt% Pt/Ce-Zr-Y mixed oxide samples were prepared, and then treated under an aging condition. The average Pt particle size measured by CO absorption method after aging was 7 nm. Redispersion treatments of the previously aged catalyst were carried out at 600 deg. C within an in-situ XAS cell in a cyclical flow of reducing/oxidizing gases. Pt L3-edge XANES spectra were collected every 1.1 second under in-situ conditions. From a change in the XANES spectra, we observed that the Pt particle size of the aged catalyst decreased from 7 to 5 nm after 60 seconds and then to 3 nm after 1000 seconds.

  3. Development of an in situ hybridization assay for the detection of ostreid herpesvirus type 1 mRNAs in the Pacific oyster, Crassostrea gigas.

    PubMed

    Corbeil, Serge; Faury, Nicole; Segarra, Amélie; Renault, Tristan

    2015-01-01

    An in situ hybridization protocol for detecting mRNAs of ostreid herpesvirus type 1 (OsHV-1) which infects Pacific oysters, Crassostrea gigas, was developed. Three RNA probes were synthesized by cloning three partial OsHV-1 genes into plasmids using three specific primer pairs, and performing a transcription in the presence of digoxigenin dUTP. The RNA probes were able to detect the virus mRNAs in paraffin sections of experimentally infected oysters 26 h post-injection. The in situ hybridization showed that the OsHV-1 mRNAs were mainly present in connective tissues in gills, mantle, adductor muscle, digestive gland and gonads. DNA detection by in situ hybridization using a DNA probe and viral DNA quantitation by real-time PCR were also performed and results were compared with those obtained using RNA probes. PMID:25455903

  4. Versatile plug flow catalytic cell for in situ transmission/fluorescence x-ray absorption fine structure measurements

    SciTech Connect

    Centomo, P.; Zecca, M.; Meneghini, C.

    2013-05-15

    A novel flow-through catalytic cell has been developed for in situ x-ray absorption spectroscopy (XAS) experiments on heterogeneous catalysts under working conditions and in the presence of a liquid and a gas phase. The apparatus allows to carry out XAS measurements in both the transmission and fluorescence modes, at moderate temperature (from RT to 50-80 Degree-Sign C) and low-medium gas pressure (up to 7-8 bars). The materials employed are compatible with several chemicals such as those involved in the direct synthesis of hydrogen peroxide (O{sub 2}, H{sub 2}, H{sub 2}O{sub 2}, methanol). The versatile design of the cell allows to fit it to different experimental setups in synchrotron radiation beamlines. It was used successfully for the first time to test nanostructured Pd catalysts during the direct synthesis of hydrogen peroxide (H{sub 2}O{sub 2}) in methanol solution from dihydrogen and dioxygen.

  5. A new airborne laser-induced fluorescence instrument for in situ detection of formaldehyde throughout the troposphere and lower stratosphere

    NASA Astrophysics Data System (ADS)

    Cazorla, M.; Wolfe, G. M.; Bailey, S. A.; Swanson, A. K.; Arkinson, H. L.; Hanisco, T. F.

    2015-02-01

    The NASA In Situ Airborne Formaldehyde (ISAF) instrument is a high-performance laser-based detector for gas-phase formaldehyde (HCHO). ISAF uses rotational-state specific laser excitation at 353 nm for laser-induced fluorescence (LIF) detection of HCHO. A number of features make ISAF ideal for airborne deployment, including (1) a compact, low-maintenance fiber laser, (2) a single-pass design for stable signal response, (3) a straightforward inlet design, and (4) a stand-alone data acquisition system. A full description of the instrument design is given, along with detailed performance characteristics. The accuracy of reported mixing ratios is ±10% based on calibration against IR and UV absorption of a primary HCHO standard. Precision at 1 Hz is typically better than 20% above 100 pptv, with uncertainty in the signal background contributing most to variability at low mixing ratios. The 1 Hz detection limit for a signal / noise ratio of 2 is 36 pptv for 10 mW of laser power, and the e fold time response at typical sample flow rates is 0.19 s. ISAF has already flown on several field missions and platforms with excellent results.

  6. In-situ detection of DNA hybridization with a microfiber Bragg grating biosensor

    NASA Astrophysics Data System (ADS)

    Sun, Dandan; Guo, Tuan; Xie, Xiaodong; Ran, Yang; Huang, Yunyun; Guan, Bai-Ou

    2014-05-01

    Microfiber Bragg gratings (mFBGs) can be used as cost-effective and relatively simple-to-implement biosensors for monitoring DNA interactions in situ. The sensors are functionalized by a monolayer of poly-L-lysine (PLL) with the specific molecular recognition probe DNA sequences to bind with high specificity to a given target. By recording the wavelength seperation between the two resonant peaks of a single mFBG, the mFBG biosensor is capable of detecting the presence of specific target DNA in situ.

  7. Determination of catecholamine in human serum by a fluorescent quenching method based on a water-soluble fluorescent conjugated polymer-enzyme hybrid system.

    PubMed

    Huang, Hui; Gao, Yuan; Shi, Fanping; Wang, Guannan; Shah, Syed Mazhar; Su, Xingguang

    2012-03-21

    In this paper, a sensitive water-soluble fluorescent conjugated polymer biosensor for catecholamine (dopamine DA, adrenaline AD and norepinephrine NE) was developed. In the presence of horse radish peroxidase (HRP) and H(2)O(2), catecholamine could be oxidized and the oxidation product of catecholamine could quench the photoluminescence (PL) intensity of poly(2,5-bis(3-sulfonatopropoxy)-1,4-phenylethynylenealt-1,4-poly(phenylene ethynylene)) (PPESO(3)). The quenching PL intensity of PPESO(3) (I(0)/I) was proportional to the concentration of DA, AD and NE in the concentration ranges of 5.0 × 10(-7) to 1.4 × 10(-4), 5.0 × 10(-6) to 5.0 × 10(-4), and 5.0 × 10(-6) to 5.0 × 10(-4) mol L(-1), respectively. The detection limit for DA, AD and NE was 1.4 × 10(-7) mol L(-1), 1.0 × 10(-6) and 1.0 × 10(-6) mol L(-1), respectively. The PPESO(3)-enzyme hybrid system based on the fluorescence quenching method was successfully applied for the determination of catecholamine in human serum samples with good accuracy and satisfactory recovery. The results were in good agreement with those provided by the HPLC-MS method. PMID:22314795

  8. Isolation and characterisation of a panel of cosmids which allows unequivocal identification of chromosome deletions involving the RB1 gene using fluorescence in situ hybridisation.

    PubMed

    Cowell, J K; Jaju, R; Kempski, H

    1994-04-01

    A series of cosmids covering the majority of the RB1 gene have been isolated from a flow sorted human chromosome 13 specific library. Using fluorescence in situ hybridisation these cosmids were all shown to hybridise to the 13q14 region but not to chromosomes known to carry subband deletions involving the RB1 gene. This panel of cosmids, therefore, can be used objectively for identification of RB1 gene deletions in tumour and normal cells. PMID:8071962

  9. Relaxation processes in hybrid organic-inorganic polymer nanosystems polymerized in situ

    PubMed Central

    2014-01-01

    The relaxation processes of hybrid organic-inorganic polymer nanosystems (OIS) synthesized by joint polymerization of organic and inorganic components were studied using methods of differential scanning calorimetry (DSC), dynamic mechanical thermal analysis (DMTA), and broadband dielectric relaxation spectroscopy (DRS). The organic component was a mixture of two products: high-molecular-weight macrodiisocyanate (MDI) with low reactivity and low-molecular-weight isocyanate-containing modifier poly(isocyanate) (PIC) with high reactivity. Sodium silicate (SS) was used as inorganic component. The structures of the OIS obtained were in the form of hybrids with covalently connected building blocks and interpenetrating networks: weakly cross-linked network MDI/SS and highly cross-linked network PIC/SS. Depending on the MDI/PIC ratio, one of the networks was prevailing and created a continuous structure with domains of second network. PACS 61.25.hk; 82.35.Lr; 64.70.pj PMID:24872804

  10. Relaxation processes in hybrid organic-inorganic polymer nanosystems polymerized in situ

    NASA Astrophysics Data System (ADS)

    Iurzhenko, Maksym; Mamunya, Yevgen; Boiteux, Gisele; Seytre, Gerard; Nikaj, Erisela; Gain, Olivier; Lebedev, Eugene; Ishchenko, Svetlana

    2014-05-01

    The relaxation processes of hybrid organic-inorganic polymer nanosystems (OIS) synthesized by joint polymerization of organic and inorganic components were studied using methods of differential scanning calorimetry (DSC), dynamic mechanical thermal analysis (DMTA), and broadband dielectric relaxation spectroscopy (DRS). The organic component was a mixture of two products: high-molecular-weight macrodiisocyanate (MDI) with low reactivity and low-molecular-weight isocyanate-containing modifier poly(isocyanate) (PIC) with high reactivity. Sodium silicate (SS) was used as inorganic component. The structures of the OIS obtained were in the form of hybrids with covalently connected building blocks and interpenetrating networks: weakly cross-linked network MDI/SS and highly cross-linked network PIC/SS. Depending on the MDI/PIC ratio, one of the networks was prevailing and created a continuous structure with domains of second network.

  11. [Molecular analysis of the triticale lines with different Vrn gene systems using microsatellite markers and hybridization in situ].

    PubMed

    Leonova, I N; Dobrovol'skaia, O B; Kminskaia, L N; Adogina, I G; Koren', L V; Khotyleva, L V; Salina, E A

    2005-09-01

    Hexaploid triticale (x Triticosecale Wittmack) lines were examined using molecular markers and the hybridization in situ technique. Triticale lines were generated based on wheat varieties differing by the Vrn gene systems and the earing times. Molecular analysis was performed using Xgwm and Xrms microsatellite markers with the known chromosomal localization in the common wheat Triticum aestivum, and rye Secale cereale genomes. Comparative molecular analysis of triticale lines and their parental forms showed that all lines contained A and B genomes of common wheat and also rye homeologous chromosomes. In the three lines the presence of D genome markers, mapped to the chromosomes 2D and 7D, was demonstrated. This was probably the consequence of the translocations of homeologous chromosomes from wheat genomes, which took part during the process of triticale formation. The data obtained by use of genomic in situ hybridization supported the data of molecular genetic analysis. In none of the lines wheat--rye translocations or recombinations were observed. These findings suggest that the change of the period between the seedling appearance and earing time in triticale lines compared to the initial wheat lines, resulted from the inhibitory effect of rye genome on wheat vernalization genes. PMID:16240635

  12. Detection of viral genomes in the liver by in situ hybridization using 35S-, bromodeoxyuridine-, and biotin-labeled probes

    SciTech Connect

    Niedobitek, G.; Finn, T.; Herbst, H.; Stein, H.

    1989-03-01

    Methods employing /sup 35/S-, biotin-, and bromodeoxyuridine (BrdUrd)-labeled DNA probes were compared for the detection of hepatitis B virus (HBV) and cytomegalovirus (CMV) in the liver. The results demonstrate that: 1) HBV can be detected reliably only by the use of radiolabeled probes, whereas methods employing nonradioactive probes obviously are not sensitive enough for this virus. The use of /sup 35/S-labeled probes shortens the exposure times considerably in comparison to tritiated probes. 2) Biotin-labeled probes are of limited value for in situ hybridization on liver tissues because the presence of endogenous avidin-binding activity often leads to false positive results. 3) Brd-Urd-labeled probes are a useful alternative to biotinylated probes for the detection of CMV. In comparison with biotinylated probes, BrdUrd-labeled probes produce a specific signal of similar staining intensity in the absence of background staining in the liver.

  13. Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

    PubMed Central

    Carter, Bradley S.; Fletcher, Jonathan S.; Thompson, Robert C.

    2010-01-01

    The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression. PMID:20699122

  14. Functionalized magnetic-fluorescent hybrid nanoparticles for cell labelling

    NASA Astrophysics Data System (ADS)

    Lou, Lei; Yu, Ke; Zhang, Zhengli; Li, Bo; Zhu, Jianzhong; Wang, Yiting; Huang, Rong; Zhu, Ziqiang

    2011-05-01

    A facile method of synthesizing 60 nm magnetic-fluorescent core-shell bifunctional nanocomposites with the ability to label cells is presented. Hydrophobic trioctylphosphine oxide (TOPO)-capped CdSe@ZnS quantum dots (QDs) were assembled on polyethyleneimine (PEI)-coated Fe3O4 nanoparticles (MNP). Polyethyleneimine was utilized for the realization of multifunction, including attaching 4 nm TOPO capped CdSe@ZnS quantum dots onto magnetite particles, altering the surface properties of quantum dots from hydrophobic to hydrophilic as well as preventing the formation of large aggregates. Results show that these water-soluble hybrid nanocomposites exhibit good colloidal stability and retain good magnetic and fluorescent properties. Because TOPO-capped QDs are assembled instead of their water-soluble equivalents, the nanocomposites are still highly luminescent with no shift in the PL peak position and present long-term fluorescence stability. Moreover, TAT peptide (GRKKRRQRRRPQ) functionalized hybrid nanoparticles were also studied due to their combined magnetic enrichment and optical detection for cell separation and rapid cell labelling. A cell viability assay revealed good biocompatibility of these hybrid nanoparticles. The potential application of the new magnetic-fluorescent nanocomposites in biological and medicine is demonstrated.

  15. Functionalized magnetic-fluorescent hybrid nanoparticles for cell labelling.

    PubMed

    Lou, Lei; Yu, Ke; Zhang, Zhengli; Li, Bo; Zhu, Jianzhong; Wang, Yiting; Huang, Rong; Zhu, Ziqiang

    2011-05-01

    A facile method of synthesizing 60 nm magnetic-fluorescent core-shell bifunctional nanocomposites with the ability to label cells is presented. Hydrophobic trioctylphosphine oxide (TOPO)-capped CdSe@ZnS quantum dots (QDs) were assembled on polyethyleneimine (PEI)-coated Fe(3)O(4) nanoparticles (MNP). Polyethyleneimine was utilized for the realization of multifunction, including attaching 4 nm TOPO capped CdSe@ZnS quantum dots onto magnetite particles, altering the surface properties of quantum dots from hydrophobic to hydrophilic as well as preventing the formation of large aggregates. Results show that these water-soluble hybrid nanocomposites exhibit good colloidal stability and retain good magnetic and fluorescent properties. Because TOPO-capped QDs are assembled instead of their water-soluble equivalents, the nanocomposites are still highly luminescent with no shift in the PL peak position and present long-term fluorescence stability. Moreover, TAT peptide (GRKKRRQRRRPQ) functionalized hybrid nanoparticles were also studied due to their combined magnetic enrichment and optical detection for cell separation and rapid cell labelling. A cell viability assay revealed good biocompatibility of these hybrid nanoparticles. The potential application of the new magnetic-fluorescent nanocomposites in biological and medicine is demonstrated. PMID:21503355

  16. Whole-mount in situ hybridization in the rotifer Brachionus plicatilis representing a basal branch of lophotrochozoans.

    PubMed

    Boell, Louis A; Bucher, Gregor

    2008-08-01

    In order to broaden the comparative scope of evolutionary developmental biology and to refine our picture of animal macroevolution, it is necessary to establish new model organisms, especially from previously underrepresented groups, like the Lophotrochozoa. We have established the culture and protocols for molecular developmental biology in the rotifer species Brachionus plicatilis Müller (Rotifera, Monogononta). Rotifers are nonsegmented animals with enigmatic basal position within the lophotrochozoans and marked by several evolutionary novelties like the wheel organ (corona), the median eye, and the nonpaired posterior foot. The expression of Bp-Pax-6 is shown using whole-mount in situ hybridization. The inexpensive easy culture and experimental tractability of Brachionus as well as the range of interesting questions to which it holds the key make it a promising addition to the "zoo" of evo-devo model organisms. PMID:18594859

  17. Homoeologous chromosome pairing between the A and B genomes of Musa spp. revealed by genomic in situ hybridization

    PubMed Central

    Jeridi, Mouna; Bakry, Frédéric; Escoute, Jacques; Fondi, Emmanuel; Carreel, Françoise; Ferchichi, Ali; D'Hont, Angélique; Rodier-Goud, Marguerite

    2011-01-01

    Background and Aims Most cooking banana and several desert bananas are interspecific triploid hybrids between Musa acuminata (A genome) and Musa balbisiana (B genome). In addition, M. balbisiana has agronomical characteristics such as resistance to biotic and abiotic stresses that could be useful to improve monospecific acuminata cultivars. To develop efficient breeding strategies for improving Musa cultivars, it is therefore important to understand the possibility of chromosome exchange between these two species. Methods A protocol was developed to prepare chromosome at meiosis metaphase I suitable for genomic in situ hybridization. A series of technical challenges were encountered, the main ones being the hardness of the cell wall and the density of the microsporocyte's cytoplasm, which hampers accessibility of the probes to the chromosomes. Key parameters in solving these problems were addition of macerozyme in the enzyme mix, the duration of digestion and temperature during the spreading phase. Results and Conclusions This method was applied to analyse chromosome pairing in metaphase from triploid interspecific cultivars, and it was clearly demonstrated that interspecific recombinations between M. acuminata and M. balbisiana chromosomes do occur and may be frequent in triploid hybrids. These results provide new insight into Musa cultivar evolution and have important implications for breeding. PMID:21835815

  18. A rapid procedure to detect in situ DNA/RNA hybrids

    SciTech Connect

    Reddy, A.R.; Sofer, W.

    1981-12-15

    A new method was developed for detecting DNA/RNA hydrids formed in situ using anti-DNA/RNA antibodies and the Peroxidase-antiperoxidase immunohistochemical procedure. Using RNA synthesized in vitro from cloned Drosophila histone genes (pDm 500H), we localized by this procedure, the histone genes to the 39 D-E region of the left arm of the second chromosome. This method has several advantages compared to conventional procedures.

  19. Laser Induced Fluorescence Emission (L.I.F.E.): In Situ Non-Destructive Detection of Microbial Life on Supraglacial Environments

    NASA Astrophysics Data System (ADS)

    Sattler, B.; Tilg, M.; Storrie-Lombardi, M.; Remias, D.; Psenner, R.

    2012-04-01

    Laser-induced fluorescence emission (L.I.F.E.) is an in situ laser scanning technique to detect photoautotrophic pigments such as phycoerythrin of an ice ecosystem such as supraglacial environments without contamination. The sensitivity of many psychrophiles to even moderate changes in temperature, and the logistical difficulties associated with either in situ analysis or sampling makes it difficult to study microbial metabolism in ice ecosystems in a high resolution. Surface communities of cold ecosystems are highly autotrophic and therefor ideal systems for L.I.F.E examinations. 532nm green lasers excite photopigments in cyanobacteria and produce multiple fluorescence signatures between 550nm and 750nm including carotenoids, phycobiliproteins which would enable a non-invasive in-situ measurement. The sensitivity of many psychrophiles to even moderate changes in temperature, and the logistical difficulties associated with either in situ analysis or sampling makes it difficult to study these cryosphere ecosystems. In general, the ice habitat has to be disrupted using techniques that usually include coring, sawing, and melting. Samples are also often chosen blindly, with little indication of probable biomass. The need for an in situ non-invasive, non-destructive technique to detect, localize, and sample cryosphere biomass in the field is therefore of considerable importance. L.I.F.E has already been tested in remote ecosystems like Antarctica (Lake Untersee, Lake Fryxell), supraglacial environments in the Kongsfjord region in the High Arctic and High Alpine glaciers but until now no calibration was set to convert the L.I.F.E. signal into pigment concentration. Here we describe the standardization for detection of Phycobiliproteins (Phycoerythrine) which are found in red algae, cyanobacteria, and cryptomonads. Similar methods are already used for detection of phytoplankton in liquid systems like oceans and lakes by NASÁs Airborne Oceanographic LIDAR since 1979. The possibility to use L.I.F.E. in ice though is a novelty and provides a promising tool to monitor vanishing ice systems like retreating glaciers.

  20. Flexible microfabricated film sensors for the in situ quantum dot-based voltammetric detection of DNA hybridization in microwells.

    PubMed

    Kokkinos, Christos; Economou, Anastasios; Speliotis, Thanasis; Petrou, Panagiota; Kakabakos, Sotirios

    2015-01-20

    A new flexible miniaturized integrated device was microfabricated for the in situ ultrasensitive voltammetric determination of DNA mutation in a microwell format, using quantum dots (QDs) labels. The integrated device consisted of thin Bi, Ag, and Pt films (serving as the working, reference, and counter electrode, respectively) deposited by sputtering on a flexible polyimide substrate. A DNA assay was employed in microwell format, where an immobilized complementary oligonucleotide probe hybridized with the biotinylated target oligonucleotide followed by reaction with streptavidin-conjugated PbS QDs. After the acidic dissolution of the QDs, the flexible sensor was rolled and inserted into the microwell and the Pb(II) released was determined in situ by anodic stripping voltammetry. Since the analysis took place directly in the microwell, the volume of the working solution was only 100 ?L and the target DNA could be detected at a concentration down to 1.1 fmol L(-1). The proposed flexible microdevice addresses the restrictions of conventional rigid electrodes while it provides a low cost integrated transducer for the ultrasensitive detection of important biomolecules. PMID:25514352

  1. Effects of central nervous system lesions on the expression of galanin: a comparative in situ hybridization and immunohistochemical study.

    PubMed Central

    Cortés, R; Villar, M J; Verhofstad, A; Hökfelt, T

    1990-01-01

    We have used in situ hybridization and immunohistochemistry to study the expression of galanin mRNA and galanin-like immunoreactivity after decortication and lesions of the ventral hippocampus. After decortication the levels of both galanin mRNA and galanin-like immunoreactivity were increased in the dorsal raphe nucleus. In addition, in decorticated rats, but not in controls, galanin mRNA could be seen in dorsal and ventral nuclei of the thalamus and in the remaining parts of the cortex. Increases in galanin mRNA and galanin-like immunoreactivity were also observed in the septum-vertical diagonal band after electrocoagulation lesions of the ventral hippocampus. In contrast, no changes were found after ibotenic acid lesions of the same hippocampal area. These results suggest that increases in the expression of galanin occur in certain neuron populations after direct lesion of their axons and/or terminal fields. Images PMID:1699231

  2. In vivo detection of porcine reproductive and respiratory syndrome virus RNA by in situ hybridization at different times postinfection.

    PubMed Central

    Sur, J H; Cooper, V L; Galeota, J A; Hesse, R A; Doster, A R; Osorio, F A

    1996-01-01

    We studied the distribution of porcine reproductive and respiratory syndrome virus (PRRSV) RNA in tissues by in situ hybridization at different times postinfection (p.i.). The probe used for in situ hybridization was prepared by reverse transcription of PRRSV RNA, followed by PCR amplification of the cDNA. The sequence amplified corresponded to 433 bp from PRRSV open reading frame 7, which is contained in the nucleocapsid protein gene and which is highly conserved in both European and American strains (H. Mardassi, L. Wilson, S. Mounir, and S. Dea, J. Clin. Microbiol. 32:2197-2203, 1994). An immunohistochemical technique was used to detect PRRSV antigen in tissue from virus-infected animals by using a monoclonal antibody specific for the PRRSV nucleocapsid protein (E.A. Nelson, J. Christopher-Hennings, T. Drew, G. Wensvoort, J.E. Collins, and D.A. Benfield, J. Clin. Microbiol. 31:3184-3189, 1993). The detection of PRRSV RNA was conducted in tissues of 6-week-old pigs that had been infected with one of three different field PRRSV isolates and collected at times ranging from 4 to 42 days p.i. Hybridization signals specific for PRRSV RNA were detected in lung, lymphoid tissues, alveolar macrophages (obtained by lavage at the time of necropsy), Peyer's patches, and kidney. The PRRSV-positive cells in these tissues appeared to be predominantly macrophages. In lung tissue we also obtained evidence suggesting the involvement of type II pneumocytes in the replication of PRRSV. During the acute period of infection there was a close correlation between the detection of RNA and the detection of nucleocapsid protein