In situ Hybridization for the Detection and Localization of the Bitter Taste Receptor Tas2r108 in the Murine Submandibular Gland

Mammals have 3 pairs of major salivary glands i.e., the parotid, submandibular, and sublingual glands. Saliva secretion of these glands is modulated by taste perception. Salivary glands are composed mainly of acinar and ductal cells. Primary saliva is secreted by acinar cells and modified during ductal flow. Recently, of the murine 35 bitter taste receptors, Tas2r108 was expressed at highest levels in the submandibular gland by qPCR. Further, Tas2r108-transfected cells respond to a range of bitter compounds, such as denatonium, quinine, colchicine, diphenidol, caffeine and dapson. The objective of the present study was to characterize the expression of Tas2r108 mRNA in acinar and/or ductal cells of the submandibular gland using in situ hybridization (ISH). Male 42-60 days old DBA2 mice were used in the study. Messenger RNAs were extracted from the submandibular gland for generating digoxigenin (DIG) labeled-cRNA probes. These probes were transcribed in anti-sense and sense orientation using T7 RNA polymerase. Dot blot hybridization was performed using DIG labeled-cRNA probes, in order to estimate integrity and optimal diluting concentration of these probes. Subsequently, ISH was performed on murine submandibular gland to detect Tas2r108 mRNA. Dot blot hybridization data demonstrated that Tas2r108 DIG labeled-cRNA anti-sense probes specifically detected Tas2r108 cDNA. ISH results showed that the anti-sense probes labeled acinar and ductal cells in the submandibular gland, whereas no staining was visible in sense controls. Interestingly, the Tas2r108 expression levels were higher in acinar than ductal cells. These results suggested that Tas2r108 might be more associated with primary saliva secretion than with ductal modification of saliva composition.

Sensitive and Specific Detection of Mycoplasma species by Consensus Polymerase Chain Reaction and Dot Blot Hybridization / 한국실험동물학회지

Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species of mycoplasmas are important pathogens that cause respiratory infection in laboratory animals and that are known to affect experimental results obtained with contaminated animals. The aim of the present study was to develop a sensitive and specific assay for the detection of mycoplasma species. To this end, we developed a polymerase chain reaction and dot blot hybridization assay (PCR/DBH) for detecting mycoplasma DNA and evaluated it for its sensitivity and specificity. Mycoplasma consensus primer pairs were used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 10(2) pg of mycoplasma purified DNA. For DBH, the amplified DNA was labeled by incorporation of digoxigenin (DIG). This DIG-labeled probe was capable of detecting 10(4) pg of purified mycoplasma DNA by DBH. PCR/DBH was more sensitive than PCR or DBH alone and was also very specific. Our PCR/DBH assay can be applied efficiently to confirm the presence of mycoplasma species on clinical samples and to differentiate between mycoplasma species infection and other bacterial infections.

Preparation of RNA probe for cd99l2 gene of zebrafish labeled with digoxingenin-UTP / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.</p><p><b>METHODS</b>The cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.</p><p><b>RESULTS</b>The plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.</p><p><b>CONCLUSION</b>The antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.</p>

Localization of herpes simplex virus type 1 DNA in latently infected BALB/c mice neurons using in situ polymerase chain reaction

Herpes simplex virus type-1 [HSV-1] establishes a lifelong latent infection in neurons following primary infection. The existence of latent HSV-1 DNA in the trigeminal ganglia of infected BALB/c mice was examined using a direct in situ PCR technique, based on Digoxigenin-11-dUTP detection system with anti-digoxigenin-peroxidase and 3,3f-diaminobenzidine [DAB] substrate. Eight-week-old male BALB/c mice were inoculated via the eye by 10[4] plaque forming unit of wild type Iranian isolates of HSV-1. After establishment of latency, trigeminal ganglia were removed and examined using in situ PCR to detect HSV-1 genome. Finally, the results of in situ PCR were verified by a two-round PCR method, using amplification cocktail of in situ reaction, as a template for a conventional gel base PCR. The results suggest that a direct in situ PCR method using a peroxidase and DAB detection system is a useful means for detection of latent HSV-1 DNA in the latently infected ganglia

Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100 percent agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.

RNA in situ hybridization for the detection of Feline Immunodeficiency Virus in infected cells.

In the present study, we have applied a novel approach to generate specific digoxigenin- and biotin-labeled RNA probes to detect Feline Immunodeficiency Virus (FIV) gag gene in the FIV-infected feline T-lymphoblastoid cell line (MYA-1). This involved cloning of the FIV gag gene into a PCR Script vector containing both T3 and T7 promoters, followed by amplification of the insert and the two promoter sequences, using specific primer sequences. The FIV RNA probes were more sensitive than FIV DNA probes. This approach should make RNA in situ hybridization more accessible for use in routine diagnosis.

Detection and Quantification of Residual Cellular DNA in the Production of Recombinant HPV-16 L1 Virus-Like Particles

A number of recombinant proteins isolated from cell sources are being produced for biopharmaceuticals. Although most biopharmaceuticals are highly purified, there is a safety concern that such recombinant products could be contaminated with impurities including adventitious virus, mycoplasma, endotoxin and oncogenic DNA. Residual DNA in recombinant biopharmaceuticals is a potential risk factor and must be evaluated and removed to meet the regulatory guidelines. Recombinant HPV type 16 L1 VLPs, recombinant protein produced in Spodoptera frugiperda (Sf) 9 insect cells, is a HPV subunit vaccine candidate which has been studied as a preventive vaccine of cervical cancers. In this study, we performed detection and quantification of residual cellular DNA in the production of recombinant HPV type 16 L1 VLPs. HPV-16 L1 VLPs were purified by processes including detergent lysis, sonication treatment, sucrose cushion centrifugation, CsCl equilibrium density centrifugation, and DNase treatment which was added to inactivate residual cellular DNA after CsCl centrifugation step. We have developed a precise assay based on a dot-blot hybridization using digoxigenin random primed labeling DNA probes for the detection and quantification of residual cellular DNA during the purification process and final products. Detection limit of residual cellular DNA was 0.1 ng in this assay and the amount of residual cellular DNA in the final product was 0.5 ng~1 ng per 100 microgram of protein. This study describes safer and more sensitive methods alternative to radioactive techniques employed for residual cellular DNA quantification of biopharmaceuticals produced by recombinant protein technology and presents method validation data demonstrating precision and reproducibility.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4 percent), followed by genotype 3 (21.4 percent), and genotype 2 (7.2 percent). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4 percent), mild (57.2 percent), and moderate (21.4 percent). Viral RNA was detected in liver cells from nine patients (64.3 percent). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Optimization of in situ hybridization assay using non-radioactive DNA probes for the detection of canine herpesvirus (CHV) in paraffin-embedded sections

Two non-radioactive probes using digoxigenin or biotin were developed for detecting canine herpesvirus (CHV) and compared for their sensitivities by in situ hybridization (ISH) in formalin fixed, paraffin embedded sections, which has been used routinely in veterinary fields. Sections of the CHV-infected cell preparation were subjected to several different ISH protocols using digoxigenin- or biotin-labeled probe respectively. Results were compared for the hybridization and background signal intensities. The best result was obtained by the optimized ISH protocol using digoxigenin-labeled probe for detection of CHV DNA. The optimized ISH assay, which developed in this study, may be a valid tool for the study of pathogenesis and diagnosis of CHV infection.

A Sensitive and Specific PCR/dot Blot Hybridization assay for the Detection of Ovine Herpesvirus 2, a Gamma Herpesvirus

Polymerase chain reaction (PCR) provides a powerful technique for identifying viruses and studying the homology between viral nucleic acids. However, PCR assay has limitations in its susceptibility to contamination or to enzymatic inhibitors. In order to avoid problems related to nucleic acid amplification, efforts have been made to obtain specific hybridization assays, such as dot blot hybridization (DBH). DBH has higher specificity and lower sensitivity than PCR. The aims of the present study were to develop a sensitive and specific assay for the detection of ovine herpesvirus 2 (OvHV-2), a gamma herpesvirus. PCR/DBH assay for detecting OvHV-2 DNA was developed and evaluated for its sensitivity and specificity. OvHV-2 specific primer pairs, 755/556, were used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 102 viral copies. For DBH, the amplified DNA with OvHV-2 specific primer pairs, 556/555, was labeled by the incorporation of digoxigenin (DIG). This DIGlabeled probe was capable of detecting 104 viral copies of purified OvHV-2 DNA by DBH. On the other hand, PCR/ DBH was more sensitive than either PCR or DBH and also very specific. The results showed that the sensitivity of PCR/DBH was higher and stronger than that of PCR and DBH alone. This PCR/DBH assay can be applied efficiently to confirm the presence of OvHV-2 virus on clinical samples and to differentiate specifically between OvHV-2 infection and other viral infections.

The in situ hybridization effort estimation between antisense RNA probe and random primed DNA probe / 法医学杂志

OBJECTIVE@#To estimate the effort between antisense RNA probe and random primed DNA probe.@*METHODS@#The in situ hybridization was conducted on parafin section from wounding model of rat skin.@*RESULTS@#Although both probes appeared positive staining, RNA probes was superior to DNA probes in terms of depth of staining and background.@*CONCLUSION@#RNA probe showed more satisfactorily on ISH.

Expression of MUC5AC and MUC8 mRNA in human nasal mucosa / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: MUC5AC is known to be a major secretory mucin in goblet cells of the mucosa of human lower respiratory tract. But in our preliminary study, we found that the levels of MUC8 mRNAs were significantly increased in the biopsy specimens of the nasal polyps whereas other mucin genes were not. This suggests the possibility that MUC8 might be one of the major overexpressed mucins in the nasal polyps. The purpose of this study is to investigate the cellular location of MUC5AC and MUC8 mRNA. Material and methods : Normal posterior ethmoid mucosa and the polyp tissue were fixed in 4% paraformaldehyde and were hybridized with the RNA riboprobe for MUC8 and the oligonucleotide probe for MUC5AC in the presence of digoxigenin (DIG). RESULT: In the normal posterior ethmoid mucosa, MUC 5AC mRNA and MUC8 mRNA were barely expressed in the epithelium and the submucosal glands. In the polyp epithelium, the expression of MUC 5AC mRNA was localized in the cytoplasm of goblet cells and the expression of MUC8 mRNA was strongly localized in the nucelus of the goblet cells, and weakly localized in the cytoplasm of the goblet cells. MUC8 mRNA was also expressed in low levels in the nucleus of the submucosal glands. CONCLUSION: MUC8 mRNA is localized mainly in the nucleus of goblet cells and is one of the major mucin genes overexpressed in goblet cells of thnasal polyp.

Improved Technique of Digoxigenin Labeled RNA in situ Hybridization

BACKGROUND: A practical RNA in situ hybridization method using digoxigenin labeled RNA probes is described in order to evaluate the technical difficulties and problems in RNA in situ hybridization. METHODS: The paraffin sections, routinely processed in the Pathology Laboratory, were tested for the possibility of RNA in situ hybridization instead of the RNase free paraffin sections, fixed in 4% paraformaldehyde and prepared using RNase protection procedures. RESULTS: Most of the paraffin sections, fixed in 10% neutral formalin solution in fresh condition, showed relatively good reaction of RNA in situ hybridization, although the necrotic tissue and autopsy specimens showed poor reaction of RNA in situ hybridization. A refixation procedure using a 4% paraformaldehyde solution was evaluated for optimal expression of mRNA in the paraffin sections. CONCLUSION: The treatment of 4% paraformaldehyde before the treatment of proteinase K showed better in situ hybridization than did the treatment of 4% paraformaldehyde after the treatment of proteinase K. Also a new Polymerase Chain Reaction (PCR)-based method of RNA probe production showed consistently good results.

TNFalpha Expression in the Paraffin-embedded Tissues of Leprosy / 대한피부과학회지

BACKGROUND: Tumor necrosis factor alpha (TNFalpha ) plays various roles in the pathogenesis of leprosy. Expression of TNFalpha according to the clinical type of leprosy has been studied in the patient serum, stimulated PBMC, and the skin lesions, but these results were controversial. Also, all studies of the skin lesions have been limited because of a small number of frozen tissues. OBJECTIVE: The purposes of this study were to estimate the availability of paraffin-embedded skin tissue of leprosy for the detection of TNFalpha mRNA and protein, to analysis the TNFalpha expression according to the clinical types of leprosy and lepra reaction, and to demonstrate various TNFalpha -positive cells in the skin lesions. METHODS:In the paraffin-embedded tissues of 17 new cases of leprosy, TNFalpha mRNA expression was detected by in situ hybridization with digoxigenin labelled oligonucleotide probe cocktail and TNFalpha protein expression by immunohistochemical stain. In addition, serum TNFalpha level was estimated by ELISA for the evaluation of the relationship between before and after lepra reaction. RESULTS: 1. The density of TNFalpha mRNA-positive cells ranged from 8 to 26 percent (mean 17 percent) and that of TNFalpha protein-positive cells ranged from 3 to 8 percent (mean 7 percent). They were the highest in borderline lepromatous leprosy lesions. The density of TNFalpha -positive cells was significantly higher by in situ hybridization than by immunohistochemical stain. 2. The TNFalpha -positive cells in paraffin-embedded skin lesion were CD8 positive lymphocytes, CD68 positive macrophages, endothelial cells, histiocytes, Schwann cells, and keratinocytes. The number of these cells before leprosy treatment was not significantly different from that after leprosy treatment. 3. The density of CD8-positive cells was significantly higher in lesions without lepra reaction than in lesions with lepra reaction, and that of CD68-positive cells was significantly higher in lepromatous leprosy lesions than in borderline lepromatous leprosy lesions. 4. Serum TNFalpha level was higher during lepra reaction than before lepra reaction and the high level continued even after resolution of lepra reaction. CONCLUSION: It suggested that the paraffin-embedded tissue in leprosy is an effective and available material for detecting expression of TNFalpha mRNA and protein. Also, in situ hybridization was more sensitive than immunohistochemical stain. The number of TNFalpha -positive cells was the highest in borderline lepromatous leprosy lesions. With further refinement, this may be an easier application of study for TNFalpha expression in leprosy.

Renal Adaptive Responses of HK alpha2 Gene(HK alpha 2a, HK alpha 2b) to the Changes of Potassium(K) Diet / 대한신장학회잡지

Recent molecular and physiological studies suggested that at least two H/K-ATPase isozymes are expressed in the rat kidney, and two distinct isoforms(HK alpha 2a, 2b) are resulted from alternative splicing of the 5-end of HK alpha 2 in distal colon by sequence analysis. Northern analysis and in situ hybridization(ISH) were carried out to analyze the expression of HK alpha 2a. and HK alpha 2b, mRNAs in rat kidney according to the changes of K-diet. Isoform specific 32P-labeled cDNA(for Northern) or digoxigenin labeled cRNA(for ISH) probes were used. Northern analysis demonstrated that HK a z. mRNA is abundantly expressed in normal(group 1: normal diet 2W) renal cortex, modestly in normal outer medulla, and weakly in normal inner medulla. The potassium-deprived rats(group 2: K-free diet 1W, and group 4: K-free diet 2W) expressed 40N lower levels in cortex and 2-4 fold higher levels of HKalpha 2a. mRNA in outer and inner medulla compared to normal rat. The potassium loading rats after potassium-deprivation(group 3: normal diet 1W after K-free diet 1W, and group 5: normal diet 1W after K-free diet 2W showed almost normal levels of HK alpha 2a. mRNA. HK alpha 2b, mRNA was not detected in any tissues of groups. By ISH, mRNA for HK alpha 2, was detected in the thick ascending limb, distal convoluted tubule, and the entire collecting duct. All groups exhibited comparable cellular patterns of labeling. Signal intensity of group 2 and 4 was less in cortical collecting duct(CCD), especially principal cells and much higher in the inner stripe of the outer medullary collecting duct(OMCDi) and the proximal inner medullary collecting duct(IMCD) compared to group 1. Group 3 and 5 exhibited signal intensity of group l. These results indicate that chronic hypokalemia enhances expression of HK alpha 2a, gene in OMCDi and proximal IMCD, decreases in CCD, and restores at normal levels in potassium-loading after potassium-deprivation and suggest that this isoform plays an important role in potassium balance by these segments accordiog to the changes of K-diet.

Restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolated from Taegu / 대한임상병리학회지

BACKGROUND: To investigate epidemiology of a specific strain, and evaluate correlation between Mycobacterium tuberculosis restriction fragment length polymorphism (RFLP) and antimicrobial susceptibility, we studied about Mycobacterium tuberculosis RFLP isolated from Taegu area. METHODS: From Oct. 1997 and Mar. 1999, we isolated 54 strains of M. tuberculosis from the patients visiting Catholic University of Taegu Hyosung, Taegu, Korea. We studied their drug susceptibility and analyzed the Pvu treated RFLP using digoxigenin labeled IS6110 probe. RESULTS: Fifty-three had more than 6 bands of RFLP and strains with 10 bands were predominant (15 strain). Cluster analysis reveals eleven distinct clusters showing 57.4% of clustered rate (31 strains from A to K) and 35 independent patterns showing 64.8% of the diversity rate at 70% similarity level. Cluster A was the largest group (7 strains) and the next was cluster B (5 strains). Most of the patients with cluster A lived in Taegu city (85.7%) and all of 2 cluster K patients lived in Euisung area. There was no correlation between RFLP pattern and antimicrobial susceptibility, but all two strains of cluster H were resistant to isoniazid. Strains of clustered were also prevalent in the people of middle class. CONCLUSIONS: Compared to the RFLP analysis in the developed countries, Korea disclosed lower rate of diversity and higher clustered patterns of M. tuberculosis. The clustered strains were also prevalent among the people of middle class.

Expression of c-erbB2 and HLA-A2 in Breast Cancer Patients / 한국유방암학회지

PURPOSE: Oncogene c-erbB2 produces a transmembrane protein similar in structure to the tyrosine kinase family. Overexpression of c-erbB2 is known to lower the survival rate of breast cancer patients. c-erbB2 protein is an important antigen for tumor specific cytotoxic T lymphocytes induction that is dependent on its presentation as stably complexed with HLA-A2. In 1997, Nistico P reported low frequency of c-erbB2 proto-oncogene overexpression in HLA A2 positive breast cancer patients. And then in this study, correlation of HLA-A2 and the c-erbB2 expression was investigated in breast cancer patients. MATERIALS AND METHODS: HLA-A DNA typing by locus-specific generic PCR and by hybridization with sequence-specific oligonucleotide probes (SSOP) was performed on peripheral blood lymphocytes from 52 breast cancer patients (a PCR-SSOP typing method, involving a PCR amplification in conjunction with digoxigenin labelled sequence-specific oligonucleotide probes). To determine c-erbB2 expression, immunohistochemistry from paraffin-embedded tissues in a series of 47 patients with available tissue blocks was performed by use of rabbit anti-human c-erbB2 oncoprotein (DAKO, Glostrup, Denmark). And then we statistically analyzed the relation between the expressions of HLA-A2 and c-erbB2 in breast cancer patients. RESULTS: 29 out of 52 patients (55.8%) were HLA-A2 positive. 23.4% (11out of 47 patients) of breast cancer patients overexpressed c-erbB2. The patients with c-erbB2 overexpression showed lower estrogen receptor positivity compared to those without c-erbB2 overexpression (10.5%, vs 33.3%). HLA-A2 positive patients showed 18.5% (5/27) of overexpression and HLA-A2 negative patients showed 30.0% (6/20) of c-erbB2 overexpression (p=0.283). CONCLUSIONS: We observed no correlation between HLA-A2 and prognostic factors in breast cancer such as tumor size, axillary nodal status. However, our results showed a tendency without statistical significance between HLA-A2 and high frequency of c-erbB2 overexpression. More accumulation of patients will be needed for better conclusions.

A Study on Prevalence of Premutation Sized FMR1 Gene Using Polymerase Chain Reaction

BACKGROUND: Fragile X syndrome is one of the most common causes of mental retardation. For its prevention, detection of premutation range CGG repeat in FMR1 gene is necessary. The aim of our study was to determine the prevalence of premutation range of CGG repeat in neonate, and to evaluate the possibility of screening test. METHODS: DNA were extracted from Guthrie paper blood spot, referred for neonatal metabolic screening test, collected during the period of March 1996 through August 1996, at Chunchon Sacred Heart Hospital. Then FMR1 gene involving CGG repeat was amplified by polymerase chain reaction, and then abnormal expansion of CGG were analyzed by agarose gel electrophoresis and digoxigenin labelled chemiluminescent detection method. RESULTS: Four cases among 669 PCR product were appeared to have abnormal CGG expansion and 3 out of the 4 cases were confirmed to have abnormal CGG repeat by chemiluminescent detection method. CONCLUSION: We found 3 premutation range CGG expansion with a prevalence of 1/233 in neonate. Although PCR based agarose gel electrophoresis alone is not suitable for screening test, it could be a useful tool for fragile X screening test in combination with chemiluminescent detection method.

Study on the Expression of Vasoactive intestinal polypeptide, Vasopressin and Oxytocin mRNAs in the Rat Brain using Double in situ Hybridization Technique / 대한해부학회지

The technique of in situ hybridization using synthetic oligonucleotides labelled by non-radioactive method was developed to localize vasoactive intestinal polypeptide, arginine-vasopressin and oxytocin mRNAs in the rat brain. Also double in situ hybridization technique where combination of non-radioactive and radioactive probes were applied was developed to localize 2 neuropeptide mRNAs in single tissue section. The results were as follows; In non-radioactive in situ hybridization methods using digoxigenin-labelled oligonucleotide probe, alkaline-phosphates method using NBT and BCIP as substrates gave the best result that specific hybridization signals were observed. In radioactive in situ hybridization methods using 35S-labelled oligonucleotide probe, specific hybridization signals were observed in both nuclear track emulsion and X-ray film autoradiography. In double in situ hybridization methods using combination of 35S-labelled and digoxigenin-labelled oligonucleotide probes, specific hybridization signals were observed in the group where K5 emulsion was applied as nuclear track emulsion. The technique of in situ hybridization using digoxigenin-labelled oligonucleotide applied in this study will be useful as alternative for radioactive in situ hybridization technique. Moreover, combination of non-radioactive and radioactive labelled probes in double in situ hybridization technique will be a useful tool for the simultaneous localization of various mRNAs in single section for the study of various neurotransmitters, neuropeptides, receptors and signal transduction molecules.