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.
Acinar Cells ; Animals ; Antisense Elements (Genetics) ; Caffeine ; Colchicine ; Digoxigenin ; DNA, Complementary ; DNA-Directed RNA Polymerases ; Humans ; In Situ Hybridization* ; Male ; Mammals ; Mice ; Quinine ; RNA, Messenger ; Saliva ; Salivary Glands ; Sublingual Gland ; Submandibular Gland* ; Taste Perception

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.
Animals ; Animals, Laboratory ; Bacteria ; Bacterial Infections ; Chimera ; Consensus ; Digoxigenin ; DNA ; Limit of Detection ; Mycoplasma ; Polymerase Chain Reaction ; Sensitivity and Specificity

OBJECTIVETo study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONThe antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.

Animals ; Central Nervous System ; embryology ; Cloning, Molecular ; Digoxigenin ; chemistry ; Gene Expression Regulation, Developmental ; Oligonucleotide Probes ; RNA Probes ; Uridine Triphosphate ; chemistry ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics

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.
Centrifugation ; Deoxyribonucleases ; Detergents ; Digoxigenin ; DNA Probes ; DNA* ; Human papillomavirus 16* ; Insects ; Limit of Detection ; Mycoplasma ; Recombinant Proteins ; Risk Factors ; Sonication ; Spodoptera ; Sucrose

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.
Animals ; Biotin/diagnostic use ; Cell Line ; DNA Probes/chemistry/genetics ; DNA, Viral/chemistry/genetics ; Digoxigenin/diagnostic use ; Dog Diseases/diagnosis/*virology ; Dogs ; Herpesviridae Infections/diagnosis/*veterinary/virology ; Herpesvirus 1, Canid/genetics/*isolation&purification ; In Situ Hybridization/methods/*veterinary ; Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity

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.
Digoxigenin ; DNA ; Hand ; Limit of Detection ; Nucleic Acids ; Polymerase Chain Reaction ; Sensitivity and Specificity

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.
Animals ; DNA, Antisense ; Digoxigenin ; Fibronectins/genetics* ; In Situ Hybridization/methods* ; RNA Probes ; RNA, Messenger/biosynthesis* ; Random Amplified Polymorphic DNA Technique/methods* ; Rats ; Rats, Sprague-Dawley ; Skin/pathology*

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.
Autopsy ; Digoxigenin* ; Endopeptidase K ; Formaldehyde ; In Situ Hybridization* ; Paraffin ; Pathology ; Polymerase Chain Reaction ; Ribonucleases ; RNA Probes ; RNA* ; RNA, Messenger

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.
Biopsy ; Cytoplasm ; Digoxigenin ; Epithelium ; Goblet Cells ; Humans* ; Mucins ; Mucous Membrane ; Nasal Mucosa* ; Nasal Polyps ; Polyps ; Respiratory System ; RNA ; RNA, Messenger*

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.
CD8-Positive T-Lymphocytes ; Digoxigenin ; Endothelial Cells ; Enzyme-Linked Immunosorbent Assay ; Histiocytes ; Humans ; In Situ Hybridization ; Keratinocytes ; Leprosy* ; Leprosy, Lepromatous ; Leprosy, Multibacillary ; Macrophages ; RNA, Messenger ; Schwann Cells ; Skin ; Tumor Necrosis Factor-alpha*