Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA extraction”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 955 records · Page 53Linked to original sources
PubMed2005

Rapid and multiplexed transcript analysis of microbial cultures using capillary electophoresis-detectable oligonucleotide probe pools.

A rapid assay for multiplex transcript analysis based on solution hybridization with pools of oligonucleotide probes was developed. In this assay called TRAC (transcript analysis with aid of affinity capture) the mRNAs to be studied are hybridized with gene-specific detection probe pools and biotinylated oligo(dT) and captured on streptavidin-coated magnetic particles. Unbound sample material and nonspecifically bound detection probes are removed and the target-specific probes are eluted and detected by capillary electrophoresis. Simultaneous treatment of 96 samples was automated using a magnetic bead particle processor. The assay enabled detection of in vitro transcribed RNA at the level of 30 amol (20 pg) and over a 300-fold linear range. Besides extracted RNA, crude cell lysates were directly used as samples. The assay was used for transcriptional analysis of selected mRNAs in the filamentous fungus Trichoderma reesei in two experimental conditions. TRAC analysis was highly reproducible, providing expression results that were consistent with conventional Northern blot analysis. The whole procedure starting from sample collecting can be carried out in 2 h, making this assay suitable for high-throughput analysis of a limited set of mRNAs e.g. in gene expression monitoring of production organism in microbial bioprocesses.

Culture Media↗
PubMed1984

Heating RNA before cell-free translation is essential for the efficient and reproducible synthesis of several peroxisomal proteins.

Total RNA, extracted with guanidinium thiocyanate from liver of clofibrate-treated rats, was translated in vitro. Heating the RNA at 60 degrees C for 5 min before translation increased the synthesis of three peroxisomal polypeptides 10-100-fold. Preproalbumin synthesis increased 10-fold. Total incorporation of [35S]methionine into proteins merely doubled. Heating is essential for reproducible and adequate translation of mRNAs coding for peroxisomal and some other proteins.

Animals↗
PubMed1978

Genomic RNA of the murine coronavirus JHM.

Genomic RNA extracted from the purified murine coronavirus JHM sediments between 52S and 54S in aqueous sucrose gradients. The RNA is single-stranded and has an apparent mol. wt. of 5.4 to 6.5 X 10(6), as determined by electrophoresis in polyacrylamide agarose gels of different concentrations. The presence of polyadenylate sequences in the RNA is demonstrated by binding to oligo-)dT) cellulose and digestion with ribonucleases A and T1. The purified RNA does not dissociate into subunits at high temperatures or in high concentrations of DMSO and is infectious.

Animals↗
PubMed1996

Mutualistic fungal endophytes express a proteinase that is homologous to proteases suspected to be important in fungal pathogenicity.

Many cultivated and wild grass species are hosts to mutualistic fungal endophytes. These associations are ecologically and agronomically significant, yet little is known regarding the physiological aspects of the interaction. In the Poa ampla/Acremonium typhinum interaction, a fungal serine proteinase, At1, is surprisingly abundant and may constitute 1 to 2% of the total leaf-sheath protein. Sequence analysis of cDNA and genomic clones indicates that proteinase At1 is a member of the eukaryotic subtilisin-like protease family. It is homologous to proteases suspected to be virulence factors in fungal pathogens of insects, nematodes, and other fungi. Gel blot analysis of RNA extracted from infected leaf-sheath tissue indicates that the proteinase At1 transcript level is extremely high. RNA gel blots and immunoblots of purified enzymes indicate that similar proteinases are produced by Epichloë festucae and Acremonium lolii, the fungal endophytes infecting Festuca rubra subsp. rubra and Lolium perenne, respectively. Fungal expression of proteinase At1-like enzymes may be a general feature of endophyte infection.

Acremonium↗
PubMed1982

Wheat Storage Proteins : ISOLATION AND CHARACTERIZATION OF THE GLIADIN MESSENGER RNAs.

A total RNA extract was prepared from developing wheat seeds using guanidine-HCl to eliminate endogenous RNase activity. The RNA preparation, substantially free of protein, carbohydrate and DNA, was chromatographed on either a poly uridylic acid-agarose or poly guanylic acid-agarose column to yield a gliadin-enriched mRNA fraction. Only slight differences were observed for the products synthesized in a wheat germ cell-free translation system when either poly adenylic acid-enriched or cytosine-rich RNA was used as a template. These results are consistent with the high proline content of the gliadins and indicate that a large proportion of the mRNA activity in these RNA preparations is directed toward gliadin synthesis. After a second affinity chromatography step, the gliadin-enriched mRNA fraction was fractionated by two cycles on sucrose-density gradient centrifugation under denaturing conditions. The RNA sedimented as a broad band with a peak at 14S and a shoulder at the 11S region of the sucrose gradient. RNA from the peak 14S fraction translated predominantly the two major gliadin polypeptides which had molecular weights of 34,000 and 36,000. Analysis of the 14S RNA by methylmercury hydroxide-agarose gel electrophoresis revealed the presence of a predominant RNA species with a molecular size of 415,000 (1,200 nucleotides).

Journal Article↗
PubMed1989

Restriction of poliovirus RNA translation in a human monocytic cell line.

The infection of the human monocytic cell line U-937 by poliovirus was characterized by a low level of virus production and a slow progression of the cytopathic effect. Infection took place in greater than 99% of the cells as revealed by a limiting dilution assay. No viral protein synthesis was evident in the infected U-937 cells when analyzed by polyacrylamide gel electrophoresis. However, a low level of poliovirus RNA translation was detected by immunofluorescence analysis using a mixture of polyclonal antisera against non-structural proteins. Although there was only a low level of viral protein synthesis, a gradual accumulation of viral mRNA took place in U-937 cells as revealed by RNA blot analysis. Similar results were obtained when the erythroleukemic cell line K-562 was used as a host cell for poliovirus. RNA extracted from infected U-937 cells was efficiently translated in rabbit reticulocyte extracts giving rise to a pattern of viral polypeptides similar to that detected when virion-purified RNA was the template used for the in vitro translation assay, suggesting that the poliovirus RNA present in infected U-937 cells is functional. The existence in U-937 cells of a discriminatory mechanism which differentially interferes with poliovirus RNA translation is discussed.

Cell Line↗
PubMed1994

Improved Method for Recovery of mRNA from Aquatic Samples and Its Application to Detection of mer Expression.

Previously described methods for extraction of mRNA from environmental samples may preclude detecting transcripts from genes that were present in low abundance in aquatic bacterial communities. By combining a boiling sodium dodecyl sulfate-diethylpyrocarbonate lysis step with acid-guanidinium extraction, we improved recovery of target mRNA from both pure cultures and environmental samples. The most significant advantage of the new protocol is that it is easily adapted to yield high recovery of mRNA from 142-mm-diameter flat filters and high-capacity cartridge filters. The lysis and extraction procedures are more rapid than previously described methods, and many samples can be handled at once. RNA extracts have been shown to be free of contaminating DNA. The lysis procedure does not damage target mRNA sequences, and mRNA can be detected from fewer than 10 bacterial cells. We used the new method to examine transcripts of genes responsible for detoxification of mercurial compounds. Induction of merA (specifying mercuric reductase) transcripts in stationary-phase Pseudomonas aeruginosa containing Tn501 occurred within 60 s of HgCl(2) addition and was proportional to the amount of Hg(II) added. The new technique also allowed the detection of merA transcripts from the microbial community of a mercury-contaminated pond (Reality Lake, Oak Ridge, Tenn.). Significant differences in merA transcript abundance were observed between different locations associated with the lake. The results indicate that the new method is simple and rapid and can be applied to the study of mer gene expression of aquatic communities in their natural habitats.

Journal Article↗
PubMed1977

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.

Chromatography, Agarose↗
PubMed1996

Detection of Borna disease virus (BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA.

In several vertebrate species, Borna disease virus (BDV), the prototype of a new group of animal viruses, causes central nervous system disease accompanied by diverse behavioral abnormalities. Seroepidemiological data indicate that BDV may contribute to the pathophysiology of certain human mental disorders. This hypothesis is further supported by the detection of both BDV antigens and BDV RNA in peripheral blood mononuclear cells (PBMCs) of patients with psychiatric disorders and the isolation of BDV from such PBMCs. Here we describe serological and molecular epidemiological studies on psychiatric patients and healthy individuals from the area of Homburg, Germany. Using a novel Western blot (immunoblot) assay, we found a BDV seroprevalence of 9.6% among 416 neuropsychiatric patients, which is significantly higher than the 1.4% found among 203 healthy control individuals. Human sera displayed a prominent immunoreactivity against the virus nucleoprotein, the p40 antigen. Reverse transcriptase-mediated PCR analysis of RNA extracted from PBMCs of a subset of 26 of the neuropsychiatric patients revealed that 50% were BDV RNA positive. Three of the 13 BDV RNA-positive patients also had BDV-positive serology, whereas one patient with serum antibodies to BDV p40 antigen did not harbor detectable BDV RNA in PBMCs. BDV p40 and p24 sequences derived from human PBMCs exhibited both a high degree of inter- and intrapatient conservation and a close genetic relationship to animal-derived BDV sequences.

Adolescent↗
PubMed1990

Relative prevalence of typical and atypical strains among rotaviruses from diarrheic pigs in conventional swine herds.

Polyacrylamide gel electrophoresis was conducted on genomic RNA extracted from rotaviruses detected in diarrheic pigs from conventional swine herds. Ninety samples contained sufficient virus for RNA band visualization and genome classification. Genome profiles were characteristic of typical group A rotaviruses in 67.8% of the 90 samples, of group B rotaviruses in 10.0%, and of group C rotaviruses in 11.1%. In 11.1% of the samples, the presence of more than 11 bands suggested concurrent infection with more than 1 strain of rotavirus. In infections among nursing pigs, 76.4% were group A rotaviruses, 7.4% were group B, 7.4% were group C, and 8.8% were coinfections. In infections among weaned pigs, 40.9% were group A, 18.2% were group B, 22.7% were group C, and 18.2% were coinfections. Coelectrophoresis with prototype OSU and Gottfried strains revealed a great diversity in electropherotype among field strains of rotavirus.

Animals↗
PubMed2002

Scarce adenylation in bacteriophage T4 mRNAs.

The degradation of mRNA is crucial for the rapid shift of bacteriophage T4 gene expression from early to late. The present study was conducted to investigate whether T4 mRNA is polyadenylated or not, because polyadenylation is known to facilitate the degradation of mRNA in Escherichia coli cells. Total RNA extracted from T4-infected cells was subjected to self-circularization or intermolecular ligation by T4 RNA ligase, and a region containing the 3'-5' junction was amplified by RT-PCR. Cloning and sequencing as well as the length distribution of amplified DNA fragments revealed no adenines at the 3'-ends of uvsY and soc RNAs. The present result suggests that T4 mRNA is not significantly adenylated.

Bacteriophage T4↗
PubMed2004

A survey of the prevalence of infectious bronchitis virus type 4/91 in Iran.

To evaluate the prevalence of infectious bronchitis virus (IBV) type 4/91 in Iran, tracheal swabs from 77 broiler flocks in 16 provinces were collected at the slaughterhouse. Swabs were subjected to RNA extraction and tested by RT-PCR, followed by a type-specific nested PCR. The viral RNA was detected in 33 samples (42.8%) from different provinces. The results indicate a relatively high prevalence of IBV type 4/91 in Iran and necessitate revising the vaccination programme against this disease.

Animals↗
PubMedDate not supplied

Altered expression of genes associated with innate immunity and inflammation in recalcitrant rhinosinusitis with polyps.

BACKGROUND: The role of the innate immune system in the pathophysiology of chronic rhinosinusitis (CRS) is poorly understood. In this study, we compared sinonasal expression of toll-like receptors (TLRs), complement components, serum amyloid A, and inflammatory genes (chemokines and cytokines) in control subjects and patients undergoing sinus surgery for CRS. METHODS: Eleven control subjects and 30 subjects with CRS unresponsive to medical management were enrolled prospectively before undergoing endoscopic sinus surgery. Ethmoid mucosal specimens were obtained surgically and processed for RNA extraction. Real-time polymerase chain reaction was used to quantitate the level of expression of messenger RNA (mRNA) for TLR, acute phase proteins, and cytokine genes. Subjects were followed for a minimum of 6 months postoperatively with nasal endoscopy to assess for recurrence of polyps. RESULTS: mRNA for all target genes was detected in the ethmoid mucosa of both control and CRS subjects. The level of gene expression was normalized to the housekeeping genes 18s RNA and glyceraldehyde-3-phosphate dehydrogenase. As compared with controls, CRS was associated with significantly higher expression of TLR2 and the inflammatory genes macrophage-inflammatory protein alpha, RANTES, and granulocyte-macrophage colony-stimulating factor. Patients with early recurrence of polyps after surgery had significantly decreased expression of TLR2, 9, and serum amyloid A and increased expression of macrophage-inflammatory protein alpha compared with surgery-responsive patients. CONCLUSION: This study shows the increased levels of expression of TLR2 and a variety of inflammatory genes in sinonasal mucosa of CRS patients compared with controls. Whether these differences play a role in pathogenesis or are merely manifestations of disease activity is worthy of investigation.

Biomarkers↗
PubMed1987

Nutritional regulation of milk protein messenger RNA concentrations in mammary acini isolated from lactating rats.

The effect of starvation, starvation and refeeding and feeding a low-protein diet on concentrations of the mRNAs for the alpha-, beta- and gamma-caseins, alpha-lactalbumin and whey acidic protein has been determined using dot-blot hybridization analyses with total RNA extracted from mammary acini isolated from lactating rats. Starvation for 48 h decreased the concentrations of all RNA species to between 5 and 20% of those for rats feeding ad libitum when expressed on a DNA basis. Refeeding for 24h restored the concentrations to control values. Consumption of a low protein diet reduced the concentrations of each mRNA by about 50%. Only minor changes were detected in the mRNA concentrations for alpha-casein, alpha-lactalbumin and whey acidic protein in samples prepared at six hourly intervals from rats receiving a restricted intake (40% ad libitum) of the control diet.

Animals↗
PubMed1986

Stimulation of delta-Aminolevulinic Acid Formation in Algal Extracts by Heterologous RNA.

Formation of the chlorophyll and heme precursor delta-aminolevulinic acid (ALA) from glutamate in soluble extracts of Chlorella vulgaris, Euglena gracilis, and Cyanidium caldarium was stimulated by addition of low molecular weight RNA derived from greening algae or plant tissue. Enzyme extracts were prepared for the ALA formation assay by high-speed centrifugation, partial RNA depletion, and gel filtration through Sephadex G-25. RNA was extracted from greening barley epicotyls, greening cucumber cotyledon chloroplasts, and growing cells of Chlorella, Euglena, Chlamydomonas reinhardtii, and Anacystis nidulans, freed of protein, and fractionated on DEAE-cellulose to yield an active component corresponding to the tRNA-containing fraction. RNA from homologous and heterologous species stimulated ALA formation when added to enzyme extracts, and the degree of stimulation was proportional to the amount of RNA added. Algal enzyme extracts were stimulated by algal RNAs interchangeably, with the exception of RNA prepared from aplastidic Euglena, which did not stimulate ALA production. RNA from greening cucumber cotyledon chloroplasts and greening barley epicotyls stimulated ALA formation in algal enzyme incubations. In contrast, tRNA from Escherichia coli, both nonspecific and glutamate-specific, as well as wheat germ, bovine liver, and yeast tRNA, failed to reconstitute ALA formation. Moreover, E. coli tRNA inhibited ALA formation by algal extracts, both in the presence and absence of added algal RNA. Chlorella extracts were capable of catalyzing aminoacyl bond formation between glutamate and both the activity reconstituting and nonreconstituting RNAs, indicating that the inability of some RNAs to stimulate ALA formation was not due to their inability to serve as glutamyl acceptors. The first step in the ALA-forming reaction sequence has been proposed to be activation of glutamate via aminoacyl bond formation with a specific tRNA, analogous to the first step in peptide bond formation. Our results suggest that the RNA that is required for ALA formation may be functionally distinct from the glutamyl-tRNA species involved in protein synthesis.

Journal Article↗
PubMed2005

RNA polymerase II blockage by cisplatin-damaged DNA. Stability and polyubiquitylation of stalled polymerase.

The consequences of human RNA polymerase II (pol II) arrest at the site of DNA damaged by cisplatin were studied in whole cells and cell extracts, with a particular focus on the stability of stalled pol II and its subsequent ubiquitylation. Site-specifically platinated DNA templates immobilized on a solid support were used to perform in vitro transcription in HeLa nuclear extracts. RNA elongation was completely blocked by a cisplatin intrastrand cross-link. The stalled polymerase was quite stable, remaining on the DNA template in nuclear extracts. The stability of pol II stalled at the site of cisplatin damage was also observed in live cells. A cell fractionation experiment using cisplatin-treated HeLa cells revealed an increased level of chromatin-associated pol II proteins following DNA damage. The stalled polymerase was transcriptionally active and capable of elongating the transcript following chemical removal of platinum from the template. Transcription inhibition by alpha-amanitin in vitro enhanced pol II ubiquitylation at ubiquitin residues Lys-6, Lys-48, and Lys-63. In live cells expressing epitope-tagged ubiquitin mutants, several ubiquitin lysines also participated in pol II ubiquitylation following DNA damage. Cisplatin treatment triggered ubiquitylation-mediated pol II degradation in HeLa cells, which could be prevented by the proteasomal inhibitor MG132. Fractionation of pol II from cells co-treated with MG132 and cisplatin indicated that the undegraded ubiquitylated polymerase was mostly unbound or only loosely associated with chromatin. These data are consistent with a model in which only a fraction of pol II, ubiquitylated in response to cisplatin damage of DNA, dissociates from the sites of platination. This altered polymerase is rapidly destroyed by proteasomes.

Cell Fractionation↗
PubMed2000

Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR.

This study describes the rapid detection of polioviruses in environmental waters by a simple reverse transcriptase-polymerase chain reaction (RT-PCR) using two primer pairs for differentiation of poliovirus from non-polio enteroviruses in a single reaction by a one-step method, combining RT and PCR in a single tube. The detection by agarose gel electrophoresis yielded 2 bands of 153-bp and 293-bp for poliovirus tested without the need for further hybridization. The detection sensitivity of this one-step duplex RT-PCR, as measured with RNA extracted by heat treatment from supernatant of infected cell extracts, was 10(-1) 50% tissue culture effective doses (TCID50). This assay was used to evaluate the ability of sample concentration by membrane filter-based adsorption and elution, and purification by a simple RNA isolation based on guanidine isothiocyanate-phenol-chloroform extraction; the system yielded a detection limit of 5 x 10(-1) TCID50 seeded in 5 liters of tap water. This protocol was applied to the poliovirus detection in environmental water collected from 2 communities in Bangkok, Thailand during February and May 1998. Of 100 samples tested, 2 water samples collected from the same open sewage pipeline at one location were positive for polioviruses and one sample collected from another sewage pipeline was positive for non-polio enterovirus while a further 97 water samples were negative for both polioviruses and non-polio enteroviruses. With poliovirus detection by cell culture technique, none of the 100 samples tested was positive for poliovirus type 1, 2 or 3. RT-PCR was more sensitive, rapid, simple and cost-effective than the cell culture technique since the two water samples which were positive for polioviruses by RT-PCR failed to be detected by cell culture. Sequence data of 293-bp amplicons from positive samples were compared with those of reference poliovirus strains in the Genbank and the EMBL databases and identity to the sequence of type 1 strain Sabin was found to be 99%.

Animals↗
PubMed1998

Quantitation of hepatitis C virus in liver and peripheral blood mononuclear cells from patients with chronic hepatitis C virus infection.

Since the natural history of hepatitis C virus-associated liver disease and the therapeutic responsiveness might vary according to liver and blood mononuclear cells viral levels, it may be important to quantitate viral RNA in liver, blood mononuclear cells and serum, and to compare these data with genotype, biochemical and histologic data. A polymerase chain reaction-based assay available for serum hepatitis C virus RNA quantitation has been optimized to quantitate viral genomes in liver and peripheral blood mononuclear cells from 47 chronic hepatitis C patients. The procedure permitted hepatitis C virus RNA quantitation in freshly isolated mononuclear cells and in total RNA extracted from frozen mononuclear cells and liver tissue. The intrahepatic viral amount (median: 2.6 x 10(3) copies/microgram RNA; range: 0 to 3.6 x 10(4) copies/microgram RNA) correlated significantly with the hepatitis C virus RNA concentration in serum (r = 0.76, P < .001), but not in mononuclear cells. Viral RNA concentrations in liver (P < .001), serum (P < 0.01) and PBMC (P < 0.05) were significantly higher in hepatitis C virus genotype 1 patients (essentially type 1b) than in non-1 type cases, but were unrelated to biochemical or histologic indexes of disease activity. In conclusion, the optimized assay permit HCV RNA quantitation in liver and peripheral blood mononuclear cells, suggesting that serum viral level is an accurate measurement of intrahepatic viral burden.

Adult↗