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At least 415 records · Page 23Linked to original sources
PubMed1983

Rapid methods for comparing the double-stranded RNA genome profiles of bluetongue virus.

Various double-stranded RNA extraction procedures, gel electrophoresis systems, and methods to detect the RNA bands in the gel were investigated to find the most rapid methods to obtain the genome profiles of bluetongue virus in small volumes (1-25 ml) of infected cell culture fluids. Rapid double-stranded RNA extraction procedures coupled with staining the acrylamide gel slabs with ethidium bromide or silver nitrate resulted in well-defined genome profiles from bluetongue virus infected cell cultures in 6-48 h. Radioactive labelling of viral RNA with 32P was time consuming, cumbersome and expensive. These techniques detect less than 0.5 micrograms of double-stranded RNA which can be obtained from one 1-ml well of a 24-well cluster plate of bluetongue virus infected cell monolayers. The methods were therefore suitable for rapid comparisons of the electropherotypes of multiple virus isolates.

Bluetongue virus↗
PubMed1980

Construction of recombinant plasmids containing rat muscle actin and myosin light chain DNA sequences.

The construction and partial characterization of recombinant bacterial plasmids carrying DNA sequences that hybridize with rat skeletal muscle actin and a myosin light chain mRNA is described. DNA of one clone hybridizes specifically with the muscle-specific alpha-actin mRNA. Three plasmid clones contain DNA inserts that hybridize with muscle as well as with nonmuscle actin mRNA. A fifth plasmid contains sequences complementary to mRNA coding for myosin light chain 2. DNA of this plasmid hybridizes specifically with RNA extracted from muscle and differentiated muscle cultures but not with RNA extracted from proliferating mononucleated myogenic cells.

Actins↗
PubMed2004

Comparison of two methods for the detection of hepatitis A virus in clam samples (Tapes spp.) by reverse transcription-nested PCR.

The detection of hepatitis A virus in shellfish by reverse transcription-nested polymerase chain reaction (RT-nested PCR) is hampered mainly by low levels of virus contamination and PCR inhibitors in shellfish. In this study, we focused on getting a rapid and sensitive processing procedure for the detection of HAV by RT-nested PCR in clam samples (Tapes spp.). Two previously developed processing methods for virus concentration in shellfish have been improved upon and compared. The first method involves acid adsorption, elution, polyethylene glycol (PEG) precipitation, chloroform extraction and PEG precipitation. The second method is based on elution with a glycine buffer at pH 10, chloroform extraction and concentration by ultracentrifugation. Final clam concentrates were processed by RNA extraction or immunomagnetic capture of viruses (IMC) before the RT-nested PCR reaction. Both methods of sample processing combined with the RNA extraction from the concentrates were very efficient when they were assayed in seeded and naturally contaminated samples. The results show that the first method was more effective in removal inhibitors and the second was simpler and faster. The IMC of HAV from clam concentrates processed by method 1 was revealed to be a very effective method of simultaneously removing residual PCR inhibitors and of concentrating the virus.

Animals↗
PubMed1975

R17 RNA replicase. VI. A large scale preparation of R17 template RNA.

A new procedure for R17 RNA preparation was devised using a 300 liters fermenter. The key factor in processing such a large quantity is the purification of phage particles prior to RNA extraction. The method involves the preparation of 250 liters of crude lysate, condensation of phage particles by the partition method, purification by DEAE-cellulose column, and removal of adherent proteins by a series of high-salt washes. The method permits preparation of approximately 3 g of phage particles free of ribosomal fragments and RNase. Phage RNA extracted in gram quantities using conventional methods often contain phenol. Thus repeated extraction of R17 RNA with salt-alcohol mixture is required.

Coliphages↗
PubMed2004

Detection of beet yellows virus by RT-PCR and immunocapture RT-PCR in Tetragonia expansa and Beta vulgaris.

Two sensitive methods, RT-PCR with phenol-extracted RNA or Triton X-100-released RNA and immunocapture RT-PCR (IR-RT-PCR) were used for the detection of Beet yellows virus (BYV) in young and old leaves of Tetragonia expansa and sugar beet (Beta vulgaris) and in sugar beet roots. Four oligonucleotide primer pairs proved suitable for the detection of BYV. The release of BYV RNA with Triton X-100 was shown to be a very effective and easy as compared to isolation of total RNA by phenol extraction with the same or higher sensitivity of subsequent PCR. Using the Triton X-100 release of RNA and IC-RT-PCR the sensitivity of detection was so high that pg amounts of BYV RNA occurring in dilutions up to 10(-6) of saps from young Tetragonia and sugar beet leaves could be detected.

Aizoaceae↗
PubMed1999

A comparison of various "housekeeping" probes for northern analysis of normal and osteoarthritic articular cartilage RNA.

Several approaches are commonly used to normalize variations in RNA loading on Northern blots, including: ethidium bromide (EthBr) fluorescence of 18S or 28S rRNA or autoradiograms of radioactive probes hybridized with constitutively expressed RNAs such as elongation factor-1alpha (ELF), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), actin, 18S or 28S rRNA, or others. However, in osteoarthritis (OA) the amount of total RNA changes significantly and none of these RNAs has been clearly demonstrated to be expressed at a constant level, so it is unclear if any of these approaches can be used reliably for normalizing RNA extracted from osteoarthritic cartilage. Total RNA was extracted from normal and osteoarthritic cartilage and assessed by EthBr fluorescence. RNA was then transferred to a nylon membrane hybridized with radioactive probes for ELF, G3PDH, Max, actin, and an oligo-dT probe. The autoradiographic signal across the six lanes of a gel was quantified by scanning densitometry. When compared on the basis of total RNA, the coefficient of variation was lowest for 28S ethidium bromide fluorescence and oligo-dT (approximately 7%), followed by 18S ethidium bromide fluorescence and G3PDH (approximately 13%). When these values were normalized to DNA concentration, the coefficient of variation exceeded 50% for all signals. Total RNA and the signals for 18S, 28S rRNA, and oligo-dT all correlated highly. These data indicate that osteoarthritic chondrocytes express similar ratios of mRNA to rRNA and mRNA to total RNA as do normal chondrocytes. Of all the "housekeeping" probes, G3PDH correlated best with the measurements of RNA. All of these "housekeeping" probes are expressed at greater levels by osteoarthritic chondrocytes when compared with normal chondrocytes. Thus, while G3PDH is satisfactory for evaluating the amount of RNA loaded, its level of expression is not the same in normal and osteoarthritic chondrocytes.

Actins↗
PubMed2000

Measuring infectious bursal disease virus RNA in blood by multiplex real-time quantitative RT-PCR.

A quantitative reverse transcription polymerase chain reaction (RT-PCR) protocol for assessing infectious bursal disease virus (IBDV) RNA levels in blood was developed using the ABI PRISM 7700 Sequence Detection System coupled with TaqMan chemistry. To control for variations in sampling and processing between samples 28S rRNA was co-amplified in a multiplex reaction and used to quantify total RNA. Relative quantification and standardisation was achieved using a log10 dilution series of RNA extracted from IBDV stock. A linear relationship was observed between input RNA and cycle threshold values (C(T)) over 5 log10 dilutions for the IBDV-specific product and 6 log10 dilutions for the 28S rRNA-specific product. As a test of the assay it was used to determine whether differences in susceptibility to IBDV observed between inbred lines of chickens could be detected at the level of viral load in the blood. Viral RNA levels peaked 2 days post-infection when there was significantly less viral RNA in the blood of resistant line 6(1) chickens compared with the more susceptible Brown Leghorns (P = 0.01). These results demonstrate that the course of IBDV infection can be monitored by quantifying IBDV RNA extracted from blood of infected chickens using TaqMan technology.

Animals↗
PubMed2000

Real-time RT-PCR fluorescent detection of tomato spotted wilt virus.

A real-time reverse transcription-polymerase chain reaction assay based on TaqMan chemistry was developed for the detection and quantification of tomato spotted wilt virus (TSWV). This method enabled sensitive, reproducible and specific detection of TSWV in 'leaf soak' and total RNA extracts from infected plants. TaqMan reliably detected TSWV in as little as 500 fg total RNA. The assay was 10-fold more sensitive than visualisation of ethidium bromide-stained bands following agarose gel electrophoresis. TSWV isolates from various crops and locations were detected with a cycle threshold of 20-26 in 1 ng total RNA extracted from fresh or freeze-dried leaves. A dilution series of in vitro transcripts from a cloned 628 base pair fragment of TSWV S RNA served as standard for quantification of viral template in infected leaf samples. The TaqMan assay detected reproducibly 1000 molecules of the target transcript.

Base Sequence↗
PubMed1992

Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation.

Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.

Acetates↗
PubMed1978

A sequential extraction method for RNA from rabbit liver.

When naphthalene-1,5-disulfonate (NDS) and phenol-cresol were used for extraction of rRNA from rabbit liver approximately 80% of the extractable RNA was recovered. By subsequent treatment of the resulting insoluble interface with 4-aminosalicylate and triisopropylnaphthalenesulfonate (PAS-TIPNS) the remaining RNA was extracted. Subcellular fractionation and polyacrylamide gel electrophoresis or sucrose density gradient analysis showed that this last fraction contrained 28s RNA from membrane-bound polysomes. Applying the sequential extraction method for isolation of poly(A)-containing mRNA showed that only a minor part of that RNA was extracted with NDS while the rest was recovered by PAS-TIPNS treatment of the interface. Thus, a purification of poly(A)-containing mRNA was possible when the sequential extraction method was used.

Aminosalicylic Acids↗
PubMed1996

Search for viral nucleic acid sequences in brain tissues of patients with schizophrenia using nested polymerase chain reaction.

BACKGROUND: We used polymerase chain reaction to search for nucleic acid sequences of several viruses in DNA and RNA extracted from brain tissues of schizophrenic and control subjects. METHODS: We extracted DNA and RNA templates from frozen brain specimens of 31 patients with schizophrenia and 23 nonschizophrenic control patients with other diseases. The extracts were subjected to polymerase chain reaction with oligonucleotide primers for 12 different viruses (cytomegalovirus, Epstein-Barr virus, herpes simplex virus type 1, human herpesvirus type 6, varicellazoster virus, measles virus, mumps virus, rubella virus, the picornavirus group, influenza A virus, human T-cell lymphotropic virus type I, and St Louis encephalitis virus), several of which have been suspected of involvement in schizophrenia. Nested primers were used to increase the sensitivity of the method. RESULTS: No amplified nucleic acid sequences encoded by the selected viral genomes were detected in extracts of any brain specimens from either schizophrenic or control patients. CONCLUSIONS: These data agree with previous studies that failed to find sequences of a number of viruses in the cerebrospinal fluid or selected areas of the brains of schizophrenic patients. Additional efforts should be undertaken to identify other known and unknown pathogens in schizophrenia, sampling more areas of the brain from subjects with a variety of clinical types of schizophrenia.

Brain↗
PubMed2005

Quantification of stem cell factor mRNA levels in the rat testis: usefulness of clusterin mRNA as a marker of the amount of mRNA of Sertoli cell origin in post pubertal rats.

Spermatogenesis is a complex cellular process regulated by gonadotrophins and local cell-cell interactions. Stem cell factor (SCF) is one of the paracrine factors, produced by the Sertoli cells, involved in the local regulation of spermatogenesis. Measurement of its testicular level is important for addressing its role in testis physiopathology. However, the relative cell composition of experimental and pathological testis samples may lead to misinterpretation in relating SCF mRNA levels to the amount of RNA extracted from the whole tissue sample. Taking into account the relative RNA content of Sertoli cell origin should provide more significant data. In the present study, three sets of experiments were intended for modifying the proportion of RNA of Sertoli cell origin in RNA extracted from whole testis tissue samples: during postnatal development; following methoxy-acetic acid (MAA) administration; and after injecting a long-acting gonadotrophin-releasing hormone agonist (GnRHa). In a first step, we demonstrated clusterin mRNA level stability in purified Sertoli cell preparations between 20 days and adulthood, and following MAA or GnRHa treatment. In a second step, we used a competitive RT-PCR assay to measure SCF and clusterin mRNA levels and expressed the amount of SCF mRNA relative to the amount of clusterin mRNA under the above experimental conditions. The SCF/clusterin mRNA level ratio was found to remain roughly stable from 20 days post-partum to adulthood; i.e. during the development of spermatogenesis. MAA administration led to an overall increase in the SCF/clusterin mRNA level ratio between 7 and 14 days after administration, consistent with the replenishment of the testis with pachytene spermatocytes and round spermatids. Conversely, after long-acting GnRHa injection, the SCF/clusterin mRNA level ratio decreased only slightly from day 21 onward. Hence, the present studies indicate that, under physiopathological conditions, the amount of clusterin mRNA is a good marker of the amount of RNA of Sertoli cell origin in testis samples at day 20 or later; different experimental alterations of spermatogenesis are associated with different patterns of SCF mRNA levels; the relationship between FSH and SCF in vivo is not as simple as that described in vitro.

Animals↗
PubMed1999

Prevention of allergic eye disease by treatment with IL-1 receptor antagonist.

PURPOSE: To determine the impact of interleukin-1 (IL-1) inhibition using IL-1 receptor antagonist (IL-1Ra) in a mouse model of allergic eye disease. METHODS: A/J mice sensitized and challenged with cat dander in the eye were treated with topical IL-1Ra or vehicle alone. Control mice were treated with IL-1Ra or vehicle but sensitized and challenged with phosphate-buffered saline alone. Immediately after the final allergen challenge, the mice were observed for behavioral changes and assessed for lid injection and chemosis. The animals were then killed, eyes and attached lids were removed for either RNA extraction or histology, and draining lymph nodes were removed for either RNA extraction or in vitro stimulation assays. Differences in chemokine message between experimental and control groups-were determined by RNase protection assays. RESULTS: Treatment with IL-1Ra in allergen-challenged animals significantly reduced allergen-induced changes in photosensitivity (60%, P = 0.0002), chemosis (50%, P = 0.0151), and injection (86.7%, P = 0.0068) compared with vehicle-treated controls. Interleukin-1Ra reduced the number of degranulated mast cells and caused a significant reduction in the number of eosinophils infiltrating the conjunctival matrix (P<0.001) after allergen challenge. Examination of chemokine mRNA taken from the conjunctiva and draining lymph nodes by RNase protection assay showed a profound decrease in the production of a number of C-C chemokines. CONCLUSIONS: These findings suggest that IL-1Ra is suppressing allergic eye disease by a down-modulation of the recruitment of eosinophils and other inflammatory cells essential for the immunopathogenesis of ocular atopy.

Allergens↗
PubMed2004

Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus.

AIM: Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes, was evaluated. METHODS: Three male patients, infected with HCV through multiple transfusions, were identified from clinical symptoms and monitored by aminotransferase for 60 months. Blood samples taken at months 0, 32, and 60 were used for viral RNA extraction. A segment of HCV NS3 region was amplified from the RNA extraction by RT-PCR and subjected to subcloning and sequencing. HLA types of these three patients were determined using complement-dependent microlymphocytotoxic assay. CTL epitopes were predicted using MHC binding motifs. RESULTS: No patient had clinical symptoms or elevation of aspartate/alanine aminotransferase. Two patients showed positive HCV PCR results at all 3 time points. The other one showed a positive HCV PCR result only at month 0. A reported HLA-A2-restricted CTL epitope had no alteration in the HLA-A2-negative carrier over 60 months. In the HLA-A2-positive individuals, all the sequences from 0 month 0 showed an amber mutation on the initial codon of the epitope. Most changes of consensus sequences in the same patient occurred on predicted cytotoxic T cell epitopes. CONCLUSION: Amber mutation and changes of consensus sequence in HCV NS3 region may be related to viral immune escape.

Adult↗
PubMed1991

RNA purification from epidermal suction blisters.

Certain inflammatory skin diseases are accompanied by increased cytokine concentrations in the epidermis. To determine whether these cytokines are synthesized in the epidermis or exported from underlying tissues, epidermal RNA was analysed for the presence of their messenger RNAs. We report a method for RNA extraction from pure epidermal samples isolated by the suction blister method. The yield of total RNA was sufficient for hybridization experiments (12-38 micrograms per seven blisters, 5 mm in diameter). Using RNA extracted by this method, we have demonstrated the presence of messenger RNA for glyceraldehyde-3-phosphate-dehydrogenase in 13 preparations from suction blisters obtained from tuberculin skin reactions, positive patch test reactions, or normal skin. We did not, however, observe messenger RNA for interleukin 1 alpha or 8 in these preparations.

Blister↗
PubMed2002

[Regulation of Smad7 gene by TGF-beta 1 in process of malignant transformation].

BACKGROUND & OBJECTIVE: Escape from transforming growth factor-beta(TGF-beta)-induced inhibition of growth and proliferation may contribute to tumorigenesis. Smad7 is inhibitory Smads of TGF-beta s signal transduction pathway and prevents TGF-beta signaling. The disorder of Smad7 may lead to the perturbation of TGF-beta signal pathway. In this study, The authors analyzed the expression of Smad7 mRNA and the regulation of Smad7 gene by TGF-beta 1 in the process of malignant transformation of BEP2D cells to investigate the mechanism of cells malignant transformation. METHODS: Cells were cultured and stimulated with TGF-beta 1 followed by RNA extraction. Purified total RNA from TGF-beta 1 treated cells and untreated controls and performed an expression analysis with a human Smad7-specific probe applying Northern blot. As a loading control for the Northern experiment, the membrane was hybridized with a human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) probe. Proteins were extracted from BEP2D and BERP35T-2 cells, then perform Western blot to examine the expression level of TGF-beta 1. RESULTS: Before stimulation with TGF-beta 1, the expression level of Smad7 in the BERP35T-2 cells were higher than that in the BEP2D cells. When stimulated with TGF-beta 1, Smad7 expression levels was upregulated evidently in BEP2D cells, but not significant in BERP35T-2 cells. The expression level of endogenetic TGF-beta 1, BERP35T-2 cells was a little higher than BEP2D cells. CONCLUSION: Over expression of Smad7 mRNA and down-regulation of the cells' responsiveness to TGF-beta 1 in human lung cancer cell line which induced by alpha-particles should be one of the mechanism of radiation induced lung cancer.

Blotting, Western↗
PubMed1976

Epstein-Barr virus-specific RNA. I. Analysis of viral RNA in cellular extracts and in the polyribosomal fraction of permissive and nonpermissive lymphoblastoid cell lines.

We analyzed the viral RNA in permissive and nonpermissive Epstein-Barr virus (EBV) Pinfected lymphoblastoid cell lines by observing the kinetics of hybridization of labeled EBV HR-1 DNA with unalabeled RNA extracted from whole cells or from the polyribosomal fraction. The data indicate the following. (i) RNA, homologous to only 3% of the EBV HR-1 DNA, is present in the polyribosomal fraction of the nonpermissive Namalwa and Kurgans cells, suggesting that the function of only a small fraction of the EBV genome is required for the expression of the EBV-related intranuclear antigen and to maintain lymphoblastoid cells in a transformed state. (ii) In general, the extent of the viral DNA sequences transcribed into stable RNA correlates with the extent of phenotypic expression of the EBV geonome. RNA extracted from virus-producing HR-1 cells contains RNA sequences transcribed from at least 45% of the viral DNA, whereas the nonpermissive cell lines contain transcripts homologous to a much smaller proportion of the EBV DNA. (iii) Viral RNA sequences found in the polyribosomal fraction of HR-1 cells arise from almost the same template as the viral RNA sequences in extracts of infractionated HR-1 cells. In contrast, in nonpermissive lymphoblastoid cells, less than 30% of the viral RNA species found in whole-cell extracts can be identified in the polyribosomal fraction. We interpret these observations to indicate that the expression of EBV genetic information is regulated in at least two ways: first, by some mechanism that regulates which DNA sequences give rise to stable RNA; second, through a mechanism whereby certain viral RBA transcripts are selectively excluded from stable association with the polyribosomes.

Antigens, Viral↗