• Title, Summary, Keyword: in situ hybridization

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Studies on In Situ Hybridization of Electron Microscopy for Detection of Viral RNA (전자현미경 In Situ Hybridization에 의한 Viral RNA의 진단에 관한 연구)

  • 최원기;주경웅;김석홍
    • Biomedical Science Letters
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    • v.2 no.2
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    • pp.257-265
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    • 1996
  • Simple stain and electron microscopic in situ hybridization is studied and applied for the identification of rabbit haemorrhagic disease viral RNA in a unicrylated preparation of the liver after innoculation of rabbit haemorrhagic disease virus. Hybridization for detection of viral RNA in unicryl embedded tissues using complementary 84 bases oligonucleotide probe labelled by biotin CE-phosphoramidite compared with 4717∼4800 sequences of rabbit haemorrhagic disease virus, modified hybridization protocol and antibiotin antibody-l0nm gold as signal marker. The best results were obtained in 0.02% glutaraldehyde, Unicryl resin cell block, biotinylated oligonucleotide probes, antibiotin-l0nm gold. In this report, RHD viral RNA was distributed widely within the mitochondria and nucleus of liver cell by electron microscopic in situ hybridization. In situ hybridization has become a standard method for localizing DNA or RNA sequences in tissue or celt preparation. In situ hybridization is detected the virus genome in the cells and tissue as specifically compared with others nucleic acid hybridization method.

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Chromosome Rearrangements Detected by Fluorescence in situ Hybridization in Human Lymphocyte Exposed to Bleomycin (Fluorescence in situ hybridization (FISH)를 이용하여 분석한 Bleomycin에 의한 사람 림프구의 염색체 재배열)

  • 손은희;정경인;정해원
    • Environmental Mutagens and Carcinogens
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    • v.17 no.1
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    • pp.12-16
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    • 1997
  • Chromosome rearrangement induced by bleomycin were identified by fluorescence in situ hybridization with probe for chromosome 4. The frequency of color junctions, translocations, dicentric and acenttic fragments increased with bleomycin dose. Different types of balanced translocation and dicentric were scored and compared. The frequency of cells exhibiting multiple aberration was higher compared to that of cells exposed to Gamma radiation suggesting that effect of bleomycin might be similar to that of high LET radiation.

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Identification of Viral RNA by Light Microscopic in situ Hybridization (광학현미경 In Situ Hybridization에 의한 Viral RNA 증명)

  • 최원기;주경웅;김석홍
    • Biomedical Science Letters
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    • v.2 no.2
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    • pp.249-255
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    • 1996
  • In this paper, a in situ hybridization(ISH) has been used to investigate the yield of viral RNA expression from each organ tissues. It is studied to establish a rapidly, specific diagnostic method detecting rabbit haemorrhagic disease virus(RHDV) RNA in 10% formalin-fixed, paraffin-em-bedded tissues of naturally RHDV-infected rabbits using oligonucleotide probe to be made by RHDV total sequences. Biotin was used as the oligonucleotide probe marker. in situ hybridization is detected the virus genome in the cells and tissue as specifically compared with others nucleic acid hybridization method. All ISH procedure of RHDV were completed to Mi-croProbe$^{TM}$ capillary action system within 1-2 hours. In this report, RHDV was distributed widely in the cytoplasm of liver cell and the cortex of kidney but lung tissue and medulla of kidney were showed to positive reaction at locally. Although not entirely free of technical limitations, nucleic acid identification holds advantages over other diagnostic tests, including exquisite sensitivity, specificity, interchangeability and speed. It is expected that, in the immediate future viral nucleic acid detection will be a prominent part of the methods used in histopathology.

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Identification of Aujeszky's disease virus by in situ hybridization (In situ hybridization 조직화학법을 이용한 오제스키병 바이러스 동정)

  • Kim, Soon-bok;Sur, Jung-hang;Moon, Oun-kyung
    • Korean Journal of Veterinary Research
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    • v.34 no.2
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    • pp.327-333
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    • 1994
  • The purpose of this study was to establish a rapid diagnostic method detecting Aujeszky's disease virus (ADV) DNA in the cultured cell monolayers (PK-15) and tissue sections of ADV(NYJ-1-87)-infected rats and pigs by in situ hybridization(ISH). Detection of specific ADV-DNA in infected cells was conducted by radiolabeled ISH method using $^{32}P-labeled $ DNA probe (BamH1 7 fragment) which contains a 6.3 Kb ADV-DNA insert. Where ADV-DNA was detected by radiolabeled ISH, the deposition of black photographic grains occurred in the nuclei and the cytoplasms of ADV-infected cells. Positive hybridization signal was often observed in the spinal trigerminal nucleus of the pons, the nucleus of the trigerminal ganglion neuron and the epithelial cells of tonsillar crypts. The results suggested that ISH is considered as a highly sensitive and reliable tool for confirmative diagnosis of this viral disease.

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Neuroanatomical Localization of Cells Containing Gonadotropin Releasing Hormone mRNA in the Brain of Frog, Rana dvbowskii, by in situ Hybridization (In situ hybridization법에 의한 북방산개구리 뇌에서 GnRH mRNA를 함유한 세포의 분포 연구)

  • 최완성;김정우
    • The Korean Journal of Zoology
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    • v.37 no.3
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    • pp.304-310
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    • 1994
  • Using in situ hybridization, we have mapped the anatomical localization of perikarya containing myNA that codes for sonadotropin releasing hormone (GnRH) in the brains of female frogs, R. dybowskii. DNA olisomers, with sequences complementary to the GnRH portion of pro-GnRH myNA sequence, were synthesized and hybridized to paraformaldehvde-fixed, sagittal sections of the whole brain stem. The distribution of the GnRH mRNA containing cell bodies was similar to that described for GnRH peptide by immunohistochemistrv. That is, cells containing GnRH mRNA were observed in the medial septal area, anterior preoptic area, ventromedial hvpothalamus and infundibular regions. However, another cell groups which contains GnRH mRNAs were also detected by in situ hybridization in the bed nucleus of hippocampal commissure, preoptic area, nucleus infundibularis dorsalis, mesencephalic nuclei and intermediolateral cell column of spinal cord areas. These results demonstrate the feasibility of using in situ hybridization as a strategy to study the distribution of GnRH neurons and the detection of GnRH gene expression in the vertebrates.

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Expression of the Multidrug Resistance Gene and its Product in Osteosarcomas of the Bone - Immunohistochemistry and In Situ Hybridization -

  • Park, Hye-Rim;Park, Yong-Koo
    • The Journal of the Korean bone and joint tumor society
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    • v.3 no.1
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    • pp.9-17
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    • 1997
  • Resistance to combination chemotherapy remains challenge in the treatment of osteosarcoma. One of the mechanisms of multiple drug resistance is an increased expression of the multidrug resistance gene(mdr1). Expression of the P-glycoprotein(mdr-1 gene product) was studied immunohistochemically and the mdr-1 gene by in situ hybridization in 33 osteosarcomas relating to various prognostic factors. Thirty cases out of 33 osteosarcomas(90.9%) showed positive cytoplasmic reactions with P-glycoprotein and nineteen instances(57.6%) were strong positive(2+). The older(>20 years) and female patients revealed more intense immunohistochemical reactions rather than those of the younger and male patients. Osteoblastic and chondroblastic osteosarcomas revealed more strong immunohistochemical reactions compared to fibroblastic types. There were no significant staining differences between the type of bony involvement, Broder's grade and the presence of necrosis. On follow-up, the mean survival rate was decreased in the strong positive group, however, this was not statistically significant. In situ hybridization for mdr-1 gene revealed positive signals in 22 cases out of 29 osteosarcomas(75.9%). Chemotherapy was done in 15 cases out of 28 patients(53.6%). The results of immunohistochemistry and in situ hybridization were not correlated with the protocols for chemotherapy. However, this result should be confirmed by a larger scale study about mdr1 mRNA expression.

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In situ PCR for the Detection of Alcelaphine Herpesvirus-l and Comparison with other Molecular Biological Diagnostic Methods (In situ PCR에 의한 alcelaphine herpesvirus-l (AHV-l)의 진단법 개발 및 다른 분자생물학적 진단법들과의 비교)

  • Kim, Ok-Jin
    • Korean Journal of Veterinary Pathology
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    • v.6 no.1
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    • pp.1-5
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    • 2002
  • A1celaphine herpesvirus 1 (AHV-1) is a causative agent of malignant catarrhal fever which is a fatal and a lymphoproliferative syndrome. AHV-1 is a gamma herpesvirus, which induces frequent latent infection and often difficult to detect its antigens or specific nucleic acids because of its low viral copies in the infected tissues. A new method, in situ PCR, is developed for the detection of AHV-1 nucleic acid in this study. Target sequences of AHV-1 open reading frame 50 gene were detected within AHV-1 infected MDBK cells. As compare with other molecular biological methods for the detection of AHV-1, in situ PCR was found to be more sensitive than in situ hybridization and to be less sensitive than nested PCR. However, nested PCR cannot afford to observe and differentiate AHV-1 infected cells. In situ PCR amplifies a target sequence within cells that can be visualized microscopically with increased sensitivity compared to detection by in situ hybridization. In situ PCR has wide applications for sensitive localization of low copy AHV-1 viral sequences within cells to investigate the role of viruses in a variety of clinical conditions and also provide the rapid, sensitive, and specific detection of AHV-1 infection.

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Digoxigenin-labeled in situ Hybridization for Diagnosis of Aujeszky's disease in Pigs (Digoxigenin-labeled in situ Hybridization을 이용한 오제스키병의 진단)

  • Kim, Soon-Bok;Moon, Oun-Kyung;Song, Geun-Suk;Yeo, Sang-Geon
    • Korean Journal of Veterinary Pathology
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    • v.1 no.2
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    • pp.119-124
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    • 1997
  • The objective of this study was to develop digoxigenin (DIG)-labeled in situ hybridization (ISH) test for diagnosis of Aujeszky's Disease(AD) in infected organs. Specific DNA with well conserved gene sequences encoding gp50 antigen in AD virus (ADV) was obtained by Polymerase Chain Reaction (PCR) method. A pair of oligonucleotide primers used in PCR allowed amplification of a 217 bp sequence from the gp50 ADV gene. The DNA was then labeled with DIG by primer labeling method for use as probe in ISH test to detect ADV nucleic acids in various tissue. Positive hybridization was demonstrated by dark pigmentation in nuclei and cytoplasm of ADV infected cells particularly in brain tonsillar crypt epithelium and pulmonary alveolar cells. This result suggests that ISH is a valuable sensitive and rapid diagnostic test for AD.

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14q32.33 Deletion Identified by array-CGH in a 5-year old-girl with Seizure

  • Cheon, Chong-Kun;Park, Sang-Jin;Choi, Ook-Hwan
    • Journal of Genetic Medicine
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    • v.8 no.1
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    • pp.62-66
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    • 2011
  • Deletions of 14q including band 14q32.33 are uncommon. Patients with terminal deletions of chromosome 14 usually share a number of clinical features. By molecular techniques (array comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH), we identified a young girl with 0.3 Mb terminal 14q32.33 deletion. Review of the nine cases with pure terminal 14q32.3 deletions described to date documented that our observation is the smallest terminal 14q deletion ever reported. The phenotype of our patient is much less severe than the phenotypes of the patients reported previously. We report our experience in examining the clinical, behavioral, and cognitive findings in a 5-year-old girl studied with chromosomal microarray hybridization and reviewed previously reported patients with 14q32 deletions.