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At least 19 recordsLinked to original sources
PubMed1992

Hepatitis C virus antibody detection by a line immunoassay and (near) full length genomic RNA detection by a new RNA-capture polymerase chain reaction.

A rapid and simple RNA-capture polymerase chain reaction assay (RCPA) for detection of hepatitis C virus (HCV) is described. The assay detects specifically the presence of (near) full length genomic RNA of HCV by capturing HCV-RNA at the 3' terminal end on magnetic beads, followed by cDNA synthesis and PCR with 5' end specific primers. Sera were obtained from 30 chimpanzees inoculated with non-A, non-B hepatitis material from various sources, 28-122 months after infection. The sera were tested for the presence of HCV-RNA by RCPA and for HCV antibodies by a Line ImmunoAssay (Inno-LIA HCV Ab). Both tests were compared and show a high degree of agreement. Screening of 30 chimpanzee sera revealed either clearing of the virus below detection level (22/30) or development of a HCV carrier state (8/30). Only 1 of 11 LIA-indeterminate samples was positive by RCPA. As the RCPA is more sensitive, it can be used to test for the presence of HCV in sera which are classified indeterminate by the LIA. The outcome of the infection seems to be independent of the nature of the inocula, suggesting that the individual immune response could determine either clearing of the virus or the development of chronic infection.

Animals↗
PubMed1992

Cardiac persistence of cardioviral RNA detected by polymerase chain reaction in a murine model of dilated cardiomyopathy.

BACKGROUND: In our model of dilated cardiomyopathy (DCM), cardiac dilatation and hypertrophy developed after inoculation of encephalomyocarditis virus (EMCV), but the infectious virus was isolated only early after infection. In this study, we investigated whether viral RNA could be detected at later times using the polymerase chain reaction (PCR). METHODS AND RESULTS: In the in vitro study, FL (human amnion) cells infected with EMCV were harvested for RNA extraction, and viral cDNA was synthesized by reverse transcription with random hexamers. Using oligonucleotide primers with homology to the 5' noncoding region of EMCV, we enzymatically amplified a 121-base pair band, which was homologous to a probe specific for EMCV as demonstrated by Southern blot hybridization. The sensitivity of this PCR technique was at the level of about 10(2)-10(3) copies of viral RNA genome. In the in vivo study, four-week-old DBA/2 mice were inoculated with EMCV intraperitoneally (10 pfu/mouse) and killed on days 1,2,3,5,7,10,14,18,28,60, and 90. The hearts were divided into three parts for purification of total RNA, histopathological examination, and to culture for infectious virus. The infectious virus was isolated from the heart after the second day but never after the 14th day. The viral genome was detectable by PCR on the second day, when very little mononuclear cell infiltration around the blood vessels was histologically visible. Positive PCR signals were observed in all hearts through day 14. Viral RNA was also detected in four of six 28-day samples, four of six 60-day samples, and two of seven 90-day samples when diffuse myocardial fibrosis was prominent, but myocardial necrosis or cellular infiltration had disappeared. CONCLUSIONS: The persistence of EMCV RNA was shown by PCR in the chronic stage of EMCV-induced myocarditis, a time when the inflammatory reaction had largely subsided. The PCR is a potentially useful method to test possible viral etiologies in idiopathic heart muscle disease or DCM.

Animals↗
PubMed1992

Unexpected heterogeneity in E2A/PBX1 fusion messenger RNA detected by the polymerase chain reaction in pediatric patients with acute lymphoblastic leukemia.

The t(1;19)(q23;p13) is the most common recurring chromosomal translocation in childhood acute lymphoblastic leukemia (ALL) and has been associated with adverse prognosis. It involves the rearrangement of two genes, PBX1 and E2A, resulting in the production of transforming chimeric DNA-binding proteins. In all previous reports in which the presence of a chimeric transcript was described, the fusion point between the coding sequences of E2A and PBX1 was found to be constant at the RNA level. We have used RNA-based polymerase chain reaction (PCR) for the detection of E2A/PBX1 messenger RNAs (mRNAs) in children with ALL at the time of diagnosis. Of 21 patients exhibiting this rearrangement, 3 (14%) expressed a variant E2A/PBX1 transcript in addition to the expected one. The relative amounts of the two chimeric mRNAs varied between the patients, but remained constant in the same patient during different stages of the disease. Sequence analysis showed an identical insertion of 27 bp at the E2A/PBX1 junction of the variant RNA species, the translation of which would result in the replacement of Val478 by 10 amino acids. The inserted sequence has not been detected in any other human transcript besides the variant E2A/PBX1 RNA species and probably represents a splicing variant of the chimeric RNA. We conclude that a subset of pediatric patients with ALL that carry the E2A/PBX1 rearrangement express two types of the chimeric mRNA. The biologic significance of this additional E2A/PBX1 transcript is discussed.

Adolescent↗
PubMed2025

Quantitative Real-Time PCR for Circular RNA Detection and Analysis.

In eukaryotes, nearly 2% of the genome represented by the coding proteins. However, emerging evidence suggest more than 75% of the human genome referred to as noncoding part also plays a crucial role in governing major regulatory pathways. Noncoding RNAs can be categorized into several groups, such as microRNAs (miRNAs), small nuclear RNA (snRNAs), small nucleolar RNA (snoRNAs), transfer RNA (tRNA), and circular RNA (circRNAs), which contribute to this regulatory landscape. Circular RNAs (circRNAs) are identified as a new class of regulatory noncoding RNAs with gene regulatory roles by acting as miRNA or RNA binding protein sponges or interacting with proteins. Researchers employ quantitative real-time PCR methods to examine circular RNA expression utilizing divergent primers for identification and quantification.

RNA, Circular↗
PubMed1991

Terminal coma affects messenger RNA detection in post mortem human temporal cortex.

In situ hybridization histochemistry has been used to study the amount of M1 muscarinic receptor mRNA in temporal cortex from subjects with Alzheimer's disease and other neurodegenerative disorders, where the duration of terminal coma was known. Total polyadenylated mRNA and glutamate decarboxylase activity were also measured. Both muscarinic receptor mRNA and enzyme activity showed a significant decline with increasing duration of terminal coma, but were not related to diagnosis. Polyadenylated mRNA signal did not show an association with coma. These data indicate the need to consider the nature of the terminal illness in post mortem studies of mRNA as well as for neurochemical research.

Alzheimer Disease↗
PubMed1991

Prediction of hepatitis C virus infectivity in seropositive Australian blood donors by supplemental immunoassays and detection of viral RNA.

The prevalence of anti-hepatitis C virus (HCV) enzyme immunoassay (EIA)-positive in 167,511 Australian volunteer blood donors from Adelaide, Melbourne, Perth, and Sydney was 0.78%. One thousand two-hundred and eighteen EIA-positive serum samples were assessed by supplemental tests including a blocking EIA and two peptide EIAs corresponding to major epitopes of the HCV C-100-3 antigen. Seven hundred and eighteen samples (59%) were negative by supplemental testing; no evidence of reactivity with other HCV gene products or HCV RNA detected by cDNA polymerase chain reaction was found in selected samples from this group. In contrast, of 43 samples randomly selected from 400 samples (32.8%) positive by supplemental testing, 88% were reactive with HCV 33-C or core antigens and 52% contained HCV RNA, suggesting contact with HCV and infectivity of most donors in this group. Most samples equivocal by supplemental testing reacted only with C-100 and not with other HCV antigens when tested by dot immunoblot assay. Only 21% had detectable HCV RNA. The battery of assays used in this study indicated that approximately 32% of HCV EIA repeatably reactive serum samples were serologically related to HCV, corresponding to a 0.25% prevalence of potentially infectious donors.

Adult↗
PubMed1992

Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction.

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.

Alanine Transaminase↗
PubMed1992

Rapid treatment of whole cells and RNA viruses for analysis of RNA by slot blot hybridization.

To avoid extensive manipulation for the purification of RNA from cells, several methods were evaluated for the direct release of RNA from influenza virus infected cells and supernatants using slot blot hybridization and non-radioactive probes. Treatment with an equal volume of 10 M aqueous guanidine hydrochloride produced the best hybridization signal. Less, but significant amounts of RNA were also released using the following treatments: dilute alkali (final concentration of 0.16 M NaOH) or 100 degrees C/5 min or RNA sample buffer containing formamide/formaldehyde, then heating at 65 degrees C/10 min. Despite the presence of large amounts of cell debris, RNA from guanidine hydrochloride treated whole cell extracts bound quantitatively to the positively charged nylon membranes. The sensitivity of RNA detection when whole cell extracts treated with guanidine hydrochloride were probed with a digoxigenin labelled cDNA probe was similar to the detection of RNA in highly purified, protein free samples. Three positively charged membranes were tested (from Amersham, ICN and Boehringer Mannheim) using two alkaline phosphatase substrates, NBT-X phos, and a chemiluminescent substrate, 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoyloxy)-phenyl- 1-1,2-dioextane (AMPPD) and a peroxidase substrate, tetramethylbenzidine (TMB). The Boehringer Mannheim membrane had the highest sensitivity for the alkaline phosphatase substrates, but the peroxidase reaction with the TMB substrate was the most consistently sensitive, irrespective of which membrane was used. The ability to quantitatively detect RNA from whole cells without any purification will allow the rapid screening of large numbers of samples for specific RNA species in research or diagnostic laboratories.

Animals↗
PubMed1992

Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients.

In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV) cDNA-PCR. From 16 of the same non-A, non-B hepatitis patients and from 5 of the same control patients formalin-fixed, paraffin-embedded liver tissue from the same biopsy was available also for HCV cDNA-PCR. In 13 of 22 non-A, non-B hepatitis patients HCV-RNA could be detected in plasma as well as in liver tissue. In the other 9 non-A, non-B hepatitis patients and in 6 control patients, no HCV-RNA was detectable in either plasma or liver tissue. The comparison between HCV cDNA-PCR results in fresh frozen versus formalin-fixed, paraffin-embedded liver biopsies showed that although detection of HCV-RNA in both correlated 100% the quantity of HCV-RNA was lower in the formalin-fixed, paraffin-embedded liver biopsies of 5 of 8 patients for whom end-point dilution titration of liver RNA was performed. We conclude that using the procedures described HCV-RNA can be reliably detected in both fresh-frozen and formalin-fixed, paraffin-embedded liver biopsies and that HCV cDNA-PCR in liver tissue may become an important assay, especially for monitoring anti-viral therapy.

Biopsy↗
PubMed1990

Flow injection analysis and real-time detection of RNA bases by surface-enhanced Raman spectroscopy.

Surface-enhanced Raman scattering (SERS) spectroscopy has been successfully interfaced with a flow injection analysis system to detect RNA bases in real time. Four of the major base components of RNA, uracil, cytosine, adenine, and guanine, were introduced into the flow injection system and were mixed with a Ag sol prior to SERS measurements. Several experimental parameters including pH, temperature, flow rate, and tubing materials were examined, and their impact on the SERS spectra is presented here. The feasibility of interfacing flow injection based SERS detection methods with liquid or high-performance liquid chromatography for the detection of individual components in a complex mixture is also assessed.

Chromatography, High Pressure Liquid↗
PubMed1990

Detection of RNA on northern blots by negative staining with aurintricarboxylic acid.

Aurintricarboxylic acid (ATA) is a well-known inhibitor of RNA and DNA modifying enzymes and was suggested as a potent RNase inhibitor for preparation of RNA (Hallick et al., 1977, Nucleic Acids Res. 4, 3055-3064). We show that ATA is a very useful stain for detecting RNA on Northern blots and slot blots although it did not fully protect purified RNA in concentrated solution against RNase A.

Animals↗
PubMed2025

Northern Blotting: Protocols for Radioactive and Nonradioactive Detection of RNA.

Northern blotting is a common technique in RNA biology, allowing to detect and quantify RNAs of interest following separation by gel electrophoresis, transfer to a membrane, and hybridization of specific anti-complementary labelled probes. In this chapter, we describe our protocol for efficient RNA extraction from yeast, separation on agarose gel, and capillary transfer to a membrane. We provide two different methods for strand-specific detection of several types of RNAs using oligonucleotide probes, the first using radioactive 32P-labelled probes, the second based on nonradioactive digoxigenin-labelled probes.

Blotting, Northern↗
PubMed1992

New data concerning the functional organization of the mammalian cell nucleolus: detection of RNA and rRNA by in situ molecular immunocytochemistry.

We have investigated the fine spatial distribution of RNA and rRNA within the Ehrlich tumor cell nucleolus by in situ hybridization with a biotin-labeled probe and by two new strategies, the polyadenylate nucleotidyl transferase-immunogold technique and immuno-labeling with anti-RNA antibodies. Besides the presence, as expected, of RNA and rRNA in the granular component and the dense fibrillar component, we show, for the first time, significant label over all the fibrillar centers of the nucleoli. When RNA and DNA were detected simultaneously on the same sections, only the fibrillar centers were positive for both. These results throw light on the controversial subject of the precise location of transcribing rRNA genes within the nucleolus. The fibrillar centers, and not the dense fibrillar component, should thus be the site of rRNA synthesis.

Animals↗
PubMed1990

Fluorescent in-situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy.

A fluorescent in-situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single-cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3-D morphology perfectly preserved. The spatial distribution of specific ribosomal RNA and messenger RNA could then be analysed by confocal microscopy of individual cells. In the hybridization procedure, biotinylated single-stranded RNA probes were used that were produced by transcription from cloned DNA fragments. Detection of poly-(A)+ RNA was performed with a poly-biotin-d(U)-tailed oligo-d(T) probe. Bound probe was detected using streptavidin-fluorescein isothiocyanate. For flow cytometry, nuclear DNA was counterstained with di-amidine-2-phenyl indol. Multi-parameter flow cytometry was used to quantify the fluorescence intensity, i.e. the hybridization signal, of thousands of cells. Confocal scanning microscopy on individual cells revealed the intracellular distribution of the target RNA. The distribution of ribosomal RNA and poly-(A)+ RNA mRNA in mouse bone marrow and the human leukaemia cell line HL60 cells was investigated. Ribosomal RNA was confined to the cytoplasm of the cells, although in many cells the nucleoli could also be distinguished. Poly-(A)+ RNA in HL60 cells was found both in the cytoplasm and nucleus. A granular cytoplasmic distribution was seen in part of these cells.

Animals↗
PubMed2025

MapID-based quantitative mapping of chemical modifications and expression of human transfer RNA.

Detection and quantification of tRNA chemical modifications are critical for understanding their regulatory functions in biology and diseases. However, tRNA-seq-based methods for modification mapping encountered challenges both experimentally (poor processivity of heavily modified tRNAs during reverse transcription or RT) and bioinformatically (frequent reads misalignment to highly similar tRNA genes). Here, we report "MapID-tRNA-seq" where we deployed an evolved reverse transcriptase (RT-1306) into tRNA-seq and developed "MapIDs" that reduce redundancy of the human tRNA genome and explicitly annotate genetic variances. RT-1306 generated robust mutations against m1A and m3C, and RT stops against multiple bulky roadblock modifications. MapID-assisted data processing enabled systematic exclusion of false-positive discoveries of modifications which arise from reads misalignment onto similar genes. We applied MapID-tRNA-seq into mapping m1A, m3C and expression levels of tRNAs in three mammary cell lines, which revealed cell-type dependent modification sites and potential translational regulation of the reduced mitochondrial activities in breast cancer.

Humans↗
PubMed1990

[Features of mutated changes of genomic RNA of cold-adapted and hr-variants of influenza group A virus, detected by RNA:RNA hybridization].

The presence of mutations in the majority of the genes of cold-adapted strains A/Leningrad/134/17/57 (H2N2), A/Leningrad/134/47/57 (H2N2) and A/PR/8/59/1 (H1N1) of influenza A virus has been demonstrated by the RNA-RNA hybridization with the subsequent electrophoresis of double-stranded RNA in 7.5% polyacrylamide gel. The strains were cultivated 17, 47 and 59 passages in the chicken embryos at 25 degrees C. In the genomes of variants passaged in chicken embryos at optimal temperature of incubation 36 degrees C (hr-variants) the used technique permits identification of a single mutant gene. The obtained data suppose the attenuation of cold-adapted vaccine strains of influenza A virus and their high genetic stability to be a result of selection of the variants obtaining multiple mutations in the genome during passaging of the virions at cold temperature. The attenuation of hr-variants is defined by 1-2 mutations (first of all in HA-gene) that makes understandable their inability to serve as donors for recombinant live influenza vaccines construction.

Antigens, Viral↗