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At least 307 records · Page 17Linked to original sources
PubMed1994

Detection of human immunodeficiency virus type 1 p24 antigen and plasma RNA: relevance to indeterminate serologic tests.

BACKGROUND: Most enzyme immunoassay-reactive specimens producing indeterminate Western blot results belong to individuals who are not infected with human immunodeficiency virus type 1 (HIV-1). However, a small percentage may correspond to early seroconversion or advanced disease, at which stage partial reactivity on Western blot may be observed. STUDY DESIGN AND METHODS: To determine the utility of HIV-1 p24 antigen and cell-free RNA detection for the resolution of Western blot-indeterminate serologic results, several types of enzyme immunoassay-positive, sero-indeterminate specimens were analyzed. Samples were obtained from infected individuals at the time of seroconversion (n = 20), from patients with AIDS (n = 2), as specimens from clinical samples obtained for diagnostic testing (n = 57), from blood donors producing persistent indeterminate results (n = 47), and from random blood donors (n = 72). RESULTS: HIV-1 p24 antigen was detected in 10 of 20 specimens collected from 9 of 12 individuals who seroconverted and in 2 of 2 AIDS patients. HIV-1 plasma RNA was positive in 22 of 22 samples from those 14 individuals. All of 57 diagnostic specimens and 47 samples obtained from persistently indeterminate donors were negative for HIV-1 p24 antigen and plasma HIV-1 RNA. One of 72 blood donor specimens was positive for HIV-1 plasma RNA and had borderline reactivity for p24 antigen. CONCLUSION: The detection of plasma RNA appears to be sensitive and specific; negative test results may be used to identify false-positive serologic reactions. The detection of p24 antigen and plasma RNA can also be used to confirm HIV-1 infection in persons with indeterminate serologic results associated with early seroconversion or late-stage disease.

Blotting, Western↗
PubMed1990

RNA probes detect nucleotide sequence homology between members of two different nairovirus serogroups.

Cloned cDNA derived from the small (S) and medium (M) genomic RNA segments of Dugbe (DUG) virus, isolate ArD44313, a member of the Nairobi sheep disease (NSD) serogroup of nairoviruses (family, Bunyaviridae) was used to prepare 32P-labelled DNA and RNA probes. The S and M segments of six isolates of DUG virus all hybridised to both DNA and RNA probes, although the M segment of isolate KT281/75 reacted only weakly. Of nine other nairoviruses tested, representing all the six other serogroups within the Nairovirus genus, none hybridised to the DNA probes. However, under conditions of low stringency, the DUG S and M RNA probes hybridised to the respective S and M segments of Ganjam (GAN) virus (another member of the NSD serogroup). The DUG S RNA probe also hybridised to the S segments of Crimean-Congo haemorrhagic fever (CCHF) virus and Hazara (HAZ) virus (members of the CCHF serogroup). The indicated sequence relationships between DUG, GAN, CCHF and HAZ viruses show that the NSD serogroup is more closely related to members of the CCHF serogroup than it is to nairoviruses of the other five serogroups.

Animals↗
PubMed2006

Prevalence of hepatitis A virus infection in Goiânia, Goiás, Brazil, by molecular and serological procedures, 1995-2002.

In this study, a total of 865 serum samples were collected between 1995 and 2002 from individuals living in Goiânia, Central Brazil, and clinically suspected of hepatitis. After exclusion of 162 samples which were positive for hepatitis B virus or hepatitis C virus, 703 samples were tested for anti-hepatitis A virus (anti-HAV) IgM antibodies by enzyme immunoassay. In addition, 588 of these samples and 22 fecal samples were analyzed by reverse transcription-nested PCR for HAV RNA detection, with positivity indices of 13.1% (77/588) and 54.5% (12/22), respectively. A similar index of viral RNA detection in anti-HAV-IgM positive or negative samples was observed in serum samples. HAV infection is a public health problem worldwide and this study underscores the extent of HAV circulation in our region.

Adolescent↗
PubMed1997

Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma.

To define the optimal blood collection parameters for plasma human immunodeficiency virus type 1 (HIV-1) viral load testing, plasma HIV-1 RNA levels were quantitated with the NASBA HIV-1 RNA QT System from blood specimens that were collected, processed, and stored under a variety of conditions that might have affected HIV-1 RNA stability. We determined that when whole blood was processed within 2 h of specimen collection the levels of HIV-1 RNA detected in EDTA-, heparin-, and acid citrate dextrose (ACD)-anticoagulated plasma samples were comparable. The levels of HIV-1 RNA in serum specimens (mean = 4.126 log units) were significantly lower (P < 0.01) than the levels in corresponding plasma samples (mean = 4.501 log units). One cycle of freeze-thaw (-70 degrees C) did not significantly reduce the level of HIV-1 RNA detected in EDTA-, heparin-, or ACD-anticoagulated plasmas. The EDTA-anticoagulated plasmas showed the smallest decrease in HIV-1 RNA copies (0.050 log units). HIV-1 RNA levels decreased over a 6-month time period in serum as well as in EDTA-, ACD-, and heparin-anticoagulated plasmas stored at -70 degrees C. However, the only significant decreases were for serum (mean decrease = 0.317 log units) and heparin-anticoagulated samples (mean decrease = 0.384 log units). A comparison of the levels of HIV-1 RNA in cell-free plasma collected in VACUTAINER EDTA Plasma Preparation Tubes and in standard VACUTAINER EDTA tubes determined that HIV-1 RNA levels were stable for up to 30 h after collection when stored at either room temperature (mean standard deviation [SD] = +/- 0.101 log units) or at 4 degrees C (mean SD = +/- 0.102 log units) as cell-free plasma or as EDTA-anticoagulated whole blood (mean SD = +/- 0.109 log units). These data indicate that EDTA-anticoagulated plasma is the most suitable and stable matrix for HIV-1 RNA quantitation.

Anticoagulants↗
PubMed1980

Binding of benzo [a]pyrene to epidermal DNA and RNA as detected by synchronous luminescence spectrometry at 77 K.

The fluorescence associated with benzo[a]pyrene [BP] moieties covalently attached to the nucleic acid (DNA plus RNA) isolated from the epidermis of BP-treated mice was examined at 77 K in frozen aqueous solutions by use of a photon-counting fluorimeter operating in the synchronous scanning mode. The excitation and emission wavelengths were scanned simultaneously with the monochromators set 28 nm apart. This setting coincides with the difference in wavelength between the excitation and emission maxima for the fluorescence of bound BP. Currently the level of detection is in the order of 1 BP residue per 200,000 bases in 40 microgram of nucleic acid. This amount of nucleic acid can be isolated from the skin of a single mouse. The method described here is generally useful for detecting the binding to DNA of nonradioactive carcinogenic polynuclear aromatic which might occur following the topical application to animal skin in vivo of complex hydrocarbon mixtures such as synthetic fuels and crude oils.

Animals↗
PubMed1997

Fast isolation of RNA to detect expression of tumor markers.

The expression status of several tumor-related proteins is of great interest in clinical examination and research. As a completion to conventional antibody staining, RT-PCR is often used today. Reliable isolation of RNA from a low number of cells is very often a critical stage of such an examination. We demonstrate here a simple and fast method to isolate RNA from only 10,000 cells and applied it to the detection of CEA, c-ERB-B2, and mdr-1 as often studied models for tumor markers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

Dexamethasone-stimulated expression of a proviral copy of mouse mammary tumor virus env mRNA.

The kinetics of dexamethasone induction of mouse mammary tumor virus RNAs in a cell line (designated line 8) which contains a deleted proviral DNA has been examined. This DNA is an apparent reverse transcription product of the env mRNA. A detectable increase in the 3.8-kilobase RNA (mouse mammary tumor virus env mRNA) was present within 15 min after hormone addition. The 2.5-kilobase RNA detected previously during hormone induction studies was also observed in these cells. Both RNAs increased in concentration up to about 4 h after hormone addition and thereafter attained the steady-state level. Pr73env was detected by immunoprecipitation and appeared to be identical to bona fide Pr73env isolated from wild-type mouse mammary tumor virus-infected cells. An additional 5.7 kilobases of RNA was detected in augmented quantities after hormone addition, but the structure of this RNA is not known.

Animals↗
PubMed1995

Detection of measles virus RNA in urine specimens from vaccine recipients.

Analysis of urine specimens by using reverse transcriptase-PCR was evaluated as a rapid assay to identify individuals infected with measles virus. For the study, daily urine samples were obtained from either 15-month-old children or young adults following measles immunization. Overall, measles virus RNA was detected in 10 of 12 children during the 2-week sampling period. In some cases, measles virus RNA was detected as early as 1 day or as late as 14 days after vaccination. Measles virus RNA was also detected in the urine samples from all four of the young adults between 1 and 13 days after vaccination. This assay will enable continued studies of the shedding and transmission of measles virus and, it is hoped, will provide a rapid means to identify measles infection, especially in mild or asymptomatic cases.

Adult↗
PubMed1985

In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.

We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.

Animals↗
PubMed1991

A simple and rapid method for the detection of RNA in formalin-fixed, paraffin-embedded tissues by PCR amplification.

A simple and rapid method for the extraction, reverse transcription and PCR amplification of RNA from formalin-fixed, paraffin-embedded tissue is described. The procedure can be completed within 24 hours. In a first application of this method we detect human albumin mRNA in liver tissue, demonstrating the feasibility to retrospectively analyze gene expression and RNA viruses in fixed tissue.

Albumins↗
PubMed1997

Cloning and characterization of partial cDNAs for woodchuck cytokines and CD3epsilon with applications for the detection of RNA expression in tissues by RT-PCR assay.

Immunologic reagents and methodology are essential to develop further the woodchuck and woodchuck hepatitis virus (WHV) as a model of immune response, inflammation, and immunotherapy in hepatitis B virus (HBV) infection. Partial cDNA clones for the woodchuck CD3epsilon marker of T cells (536 bp) and for selected woodchuck cytokines were developed, including IL-1beta (332 bp), IL-2 (249 bp), IL-4 (205 bp), IL-10 (476 bp), IFN-gamma (476 bp), and TNF-alpha (381 bp). This panel of markers includes sets to measure RNAs for T cells (CD3epsilon), immune response induction (IL-1beta, IL-2), TH subsets (TH1, IL-2/IFN-gamma vs. TH2, IL-4/IL-10), and effector molecules that regulate hepadnavirus replication and liver injury (IFN-gamma, TNF-alpha). Primers representing highly conserved segments of genes from other species were used to derive the partial cDNA clones. Target RNA was obtained from woodchuck peripheral blood mononuclear cells (PBMC) that were stimulated in vitro with ConA, LPS, and human rIL-2. The cDNA clones were validated by 1) comparison with other species for homologies in the nucleotide and predicted amino acid sequences and 2) a first generation assay demonstrating induction of the respective RT-PCR products in stimulated woodchuck PBMC. The corresponding RNAs were also detectable in most cases in the total RNA from the livers of uninfected and WHV-infected woodchucks and differential expression of IFN-gamma and TNF-alpha RNAs was suggested. Second generation, semi-quantitative assays for the RNAs were validated using RT-PCR and dot-blot hybridization with 32P-oligomers derived from the internal sequences of the respective clones. Continued study of the woodchuck immune response to WHV infection using these assays will provide insight into the kinetics and immune mechanisms that initiate and maintain chronic hepadnavirus infection and, hence, enable development of improved immunotherapies for established chronic HBV infection.

Amino Acid Sequence↗
PubMed2001

Detection of HGV RNA in digestive organs.

The organ where the GB virus (GBV)-C/hepatitis G virus (HGV) localizes and proliferates is not known. We examined the digestive organs for HGV RNA to determine the localization of the HGV. Two cases of patients with serum-positive HGV RNA were investigated. We embedded surgically excised materials and digestive secretion materials from cases 1 and 2 in paraffin blocks. The tissue specimens investigated included lymph nodes No. 201 and 202, ascending colon (nontumor and tumor area), ileocecum, appendix, liver (nontumor and tumor area) and gall bladder. We made cDNA after extraction of total RNA from thin tissue sections and detected HGV RNA with a reverse transcription polymerase chain reaction method. No HGV RNA was detected in liver, colon and gall bladder tissues. HGV RNA was only detected in the appendix tissue. Comparison of nucleotide sequences of PCR products from serum and appendix was almost the same. Homology between US type (PNF2161) and the serum and appendix PCR products was 92.6 and 93.6%, respectively. These results suggest that HGV proliferates in the appendix and is carried by the portal blood flow to the liver, and may cause a hepatitis reaction in the liver.

Aged↗
PubMed2003

Presence of HCV RNA in hepatic tissue and effectiveness of treatment with interferon and ribavirin.

BACKGROUND: The aim of the study was to assess the prognostic value of HCV RNA detection in hepatic tissue for the response to treatment with interferon alfa (IFN) and ribavirin (RIB) in patients with chronic hepatitis C. PATIENTS AND METHODS: A group of 55 patients infected with HCV was examined before the commencement of treatment with IFN and RIB. In all of them HCV RNA was detected in the serum. The presence of HCV RNA in hepatic tissue was confirmed using a technique combining a new method of HCV RNA extraction from hepatic tissue and detection of HCV RNA with RT-PCR Cobas Amplicor Roche tests. The group of 55 patients received IFN (3-6 MU, 3 times a week) and RIB (800-1200 mg daily) for 6 to 12 months. The response to treatment was assessed 6 months after completion of the therapy. Sustained response (SR) was defined as absence of HCV RNA in the serum and normalization of ALT levels. RESULTS: The presence of HCV RNA in hepatic tissue was confirmed in 48 patients (group A), and in 7 (group B) HCV RNA was not detected in the liver. SR was observed in 17 patients from group A and 6 from group B. Significant correlation (p < 0.05) was found between detection of HCV RNA in hepatic tissue and response to IFN + RIB therapy. CONCLUSIONS: As it follows from our results, the detection of HCV RNA in the hepatic tissue of a patient with chronic hepatitis C may be a negative prognostic factor for the patient's response to IFN + RIB therapy.

Adolescent↗
PubMedDate not supplied

[Turnover of nuclear RNA. III. Detection and characterization of superstable molecules].

In prolonged experiments in which rats were injected with 14C-orotic acid for 7-10 days with subsequent measurements (during 1-2 months) of the label in various rat liver RNA fractions and in the acid-soluble pool, a superstable nuclear RNA fraction was found (T1/2-4.2 days in quiescent rat liver and 4.3 days in regenerating rat liver). A small fraction of this RNA (5-10% of total) is polyadenylated and heterogeneously distributed when electrophoresed in 1% agarose under partially denaturing conditions. Using the conveyer model of synthesis and processing of nuclear RNA advanced earlier the spectrum of turnover rates of superstable transcripts was determined. The data obtained are discussed in light of the hypothesis on posttranscriptional regulation of gene expression.

Animals↗
PubMed2004

Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation.

By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with approximately 1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with approximately 5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.

DNA↗