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 793 records · Page 44Linked to original sources
PubMed1985

Identification of a 60-kDa phosphoprotein that binds stored messenger RNA of Xenopus oocytes.

Rapidly labelled, polyadenylated RNA is contained in three distinct fractions isolated from homogenized amphibian oocytes: (a) in ribonucleoprotein particles that are associated with a fibrillar matrix, the complexes sedimenting at greater than 1500S; (b) in ribonucleoprotein particles that sediment at 20-120S and have the characteristics of stored (maternal) messenger ribonucleoprotein (mRNP) and (c) in polyribosomes that sediment at 120-360S. We have compared the RNA and protein components of the first two of these RNP fractions. The polyadenylated RNA extracted from the two RNP fractions differs in that the RNA from fibril-associated RNP contains a much higher content of repeat sequences than does the RNA from mRNP. In other words, the RNA from fibril-associated RNP is largely unprocessed and may constitute a premessenger state, which for convenience is referred to as premessenger RNP (pre-mRNP). RNA-binding experiments demonstrate that the polypeptide most tightly bound in pre-mRNP is a 54-kDa component (p54), whereas the polypeptide most tightly bound in mRNP is a 60-kDa component (p60). Antibodies raised against p60 are used to show that this polypeptide is a common major component of pre-mRNP and mRNP and that it is also located in oocyte nuclei. However the state of p60 is modified between the premessenger and stored message levels: the polypeptide in mRNP is heavily phosphorylated whereas the equivalent polypeptide in pre-mRNP is completely unphosphorylated. The relative roles of the presence of repeat sequences and phosphorylation of mRNA-associated protein in blocking translation are discussed.

Animals↗
PubMed1995

Capped nonviral sequences at the 5' end of the mRNAs of rice hoja blanca virus RNA4.

Subgenomic RNAs of both polarities corresponding to rice hoja blanca virus (RHBV) ambisense RNA4 were detected in RHBV-infected rice tissues. Total RNA extracted from RHBV-infected and noninfected rice tissues and RNA4 purified from RHBV ribonucleoprotein particles were used as templates for primer extension studies. The RNAs extracted from RHBV-infected tissues contain a population of RNA molecules with 10 to 17 nonviral nucleotides at their 5' end. The RNA-cDNA hybrids resulting from primer extension of such RNA molecules were specifically immunoselected with anti-cap antibodies, indicating that the subgenomic RNAs are capped and probably serve as mRNAs and that the additional nucleotides at their 5' end possibly derive from host mRNAs via a cap-snatching mechanism.

Base Sequence↗
PubMed1983

A high molecular weight precursor of luteinizing hormone releasing hormone from rat hypothalamus.

A poly A+ enriched fraction of total RNA extracted from rat hypothalamus was translated in a messenger RNA-dependent cell free system from rabbit reticulocyte lysate supplemented with 3H-leucine. The translation products were immunoprecipitated using a specific antiserum to luteinizing hormone releasing hormone (LHRH). Following sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography of the immunoprecipitates, a single immunoreactive polypeptide was detected with an apparent molecular weight of 28,000. This is approximately twenty times larger than the active peptide, and thus suggests the presence of a high molecular weight precursor for the decapeptide, LHRH.

Animals↗
PubMed1996

Paradoxical effects of overexpression of the type I insulin-like growth factor (IGF) receptor on the responsiveness of human breast cancer cells to IGFs and estradiol.

Estrogens increase the proliferative response of estrogen-responsive breast cancer cells to insulin-like growth factors (IGFs). The mechanisms involved are unclear, but the observation that estradiol increases type I IGF receptor levels in MCF-7 breast cancer cells has suggested that the increased response may be due to increased expression of type I IGF receptor. The purpose of this study was to investigate this hypothesis by using a retroviral expression vector to constitutively over-express the type I IGF receptor in estrogen-responsive breast cancer cells. We isolated clones of infected MCF-7 cells that expressed up to 4.5-fold more receptor than the estradiol-induced level in cells infected with a control vector. Hybridization of a type I IGF receptor complementary DNA probe to RNA extracted from these clones showed that most of the receptor RNA was transcribed from the retroviral provirus. Estrogen receptor continued to be expressed in clones overexpressing type I IGF receptor, and overexpression had little effect on the induction of an estrogen-regulated gene by estradiol and the proliferative response to IGFs alone or estradiol alone. Overexpression did, however, alter the proliferative response to IGFs in the presence of estradiol. The three clones analyzed showed an increased sensitivity to low IGF-I concentrations and a paradoxical attenuation of the synergistic effect between estradiol and IGF-I at high IGF-I concentrations. Collectively, these experiments show that the level of expression of the type I IGF receptor is an important determinant in the responsiveness of breast cancer cells to estrogen, but the observation that the response of cells to estradiol alone is not affected by constitutive overexpression of the type I IGF receptor suggests that estrogens stimulate the proliferation of breast cancer cells by regulating the expression of genes in addition to the type I IGF receptor.

Breast Neoplasms↗
PubMed2006

Targeted inhibition of p57 and p15 blocks transforming growth factor beta-inhibited proliferation of primary cultured human limbal epithelial cells.

PURPOSE: To evaluate the role of cyclin-dependent kinase inhibitors p57 and p15 in transforming growth factor (TGF)-beta1 or TGF-beta2 inhibited proliferation of primary cultured human limbal epithelial cells using short interfering RNA (siRNA). METHODS: Primary cultured human limbal epithelial cells were treated with TGF-beta1 or TGF-beta2 for 6 and 24 h, and total RNA extracted for RT-PCR and real-time PCR using primers for p21, p27, and p57 (CipP/Kip family) and p15 and p19 (INK4 family). Proteins were extracted for western blot analysis of p57 and p15. For RNA interference, primary cultured human limbal epithelial cells were transfected with annealed double-stranded siRNA (67 nM) specific for p57, p15, or siRNA-Fluorescein (siRNA-F; as a negative control) followed by treatment with TGF-beta1 or TGF-beta2 at 1 ng/ml. P57 and p15 were quantitatively detected by real-time PCR and western blot; and immunolocalized by immunofluorescent staining. The effects of TGF-beta1 or TGF-beta2 on cell proliferation were evaluated by BrdU incorporation and MTT assay. RESULTS: TGF-beta1 or TGF-beta2 significantly inhibited primary cultured human limbal epithelial cell proliferation measured by BrdU incorporation and MTT assay. TGF-beta1 or TGF-beta2 upregulated the expression of p57 and p15 mRNA and protein, but did not effect the expression of p19, p21, or p27. The siRNA transfection efficiency of these cells was 75% and no cellular toxicity was observed by 24 h. The TGF-beta1 or TGF-beta2 stimulated expression of p57 and p15 mRNA were markedly blocked by siRNA-p57 or siRNA-p15, respectively, but not by siRNA-F. The TGF-beta1 or TGF-beta2 suppression of epithelial proliferation measured by BrdU incorporation and MTT generation was increased to near normal levels by siRNA-p57 or siRNA-p15. Western blot and immunofluorescent staining showed that levels of p57 and p15 proteins were equally reduced in the cytoplasm and nucleus. CONCLUSIONS: These findings demonstrate that TGF-beta1 and/or TGF-beta2 inhibit proliferation of primary cultured human limbal epithelial cells and that p57 and p15 play roles in this process.

Cell Proliferation↗
PubMedDate not supplied

HCV and lymphoproliferative diseases.

METHODS: Genomic and replicative forms of HCV-RNA in B lymphocytes were detected by RT-PCR, and HCV genotyping was performed using universal and type-specific primers for the core region. Immunoglobulin gene rearrangement was detected by RT-PCR. RESULTS: The presence of genomic and replicative forms of HCV-RNA in the 5'NC region was investigated on total RNA extracted from subpopulations of PBMC. The frequency of HCV-RNA was higher in the B lymphocytes than in other PBMC. In two patients a larger sized band was present in the B lymphocytes and PMN; this band could represent either another form of HCV-RNA or a cross-reaction between cellular RNA and HCV primers. HCV-RNA detected using primers for the core region was negative in the patients examined. Immunoglobulin monoclonal gene rearrangement was present on the cDNA in all of the HCV and type II cryoglobulinemia positive samples except two; in contrast, it was absent in the HCV positive and cryoglobulinemia negative samples. The analysis of immunoglobulin monoclonal gene rearrangement on DNA showed the presence of new positive samples among the HCV positive, type II cryoglobulinemia negative patients, who had been negative when PCR was performed on cDNA. Denaturing sequencing gel showed clearer results than agarose gel. CONCLUSIONS: The early detection of immunoglobulin monoclonal gene rearrangement and expression is very important because it could provide evidence of the possible lymphoproliferative evolution of HCV infection. In addition, these investigations together with PCR product sequencing could show us the steps in the clonal selection of B lymphocytes towards malignant transformation, in which HCV plays a direct and/or indirect role.

Base Sequence↗
PubMed1996

Coxsackievirus B3 genomes detected by polymerase chain reaction: evidence of latent persistency in the myocardium in experimental murine myocarditis.

We have investigated the time course after infection in coxsackievirus B3 murine myocarditis to determine the extent (if any) of persistent or latent infection that might be responsible for recurrence. We employed a polymerase chain reaction (PCR) method that can detect an extremely small amount of genome by amplification techniques in four-week-old BALD/c mice (n = 140) infected with coxsackievirus B3 by a single intraperitoneal injection of 1 x 10(4) plaque-forming units (PFU)/mouse (Group 1) and 1 x 10(2) PFU/mouse (Group 2). Mice were sacrificed on days 3, 5, 7, 10, 14, 21 and 28, and their hearts were resected for RNA extraction. Single chain DNA was synthesized from 1 microgram of RNA and the viral genome was amplified by PCR. The virus genome was strongly amplified in Group 1 from days 3 to 10, and in Group 2 from days 5 to 7, but afterwards both amplifications rapidly diminished. However, a positive signal, though very faint, persisted in both groups until day 28, by which time all histological evidence of myocarditis had disappeared in Group 2. Our results demonstrated that there was persistent or latent virus infection in the myocardium throughout the entire study period of 28 days. Such persistence might provide a pathomechanism for the exacerbation and recurrence of myocarditis.

Animals↗
PubMed2002

Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.

Flavonoids↗
PubMed1979

Multiparameter kinetic analysis of killer cell initiation by using immune RNA.

By treating nonsensitized C57BL/6J spleen derived lymphocytes with EL-4 tumor cell directed xenogeneic extracted RNA we were able to monitor early changes in cellular DNA content by flow cytometric (FCM) analysis and 3H-thymidine uptake. These kinetic parameters were correlated with cell mediated cytotoxicity which appeared as early as 8 hr after activation as measured by release of chromium-51 from labeled EL-4 target cells. Flow cytometric analysis and 3H-thymidine uptake data shown peak S phase activity at 72 hr. Maximum cytotoxicity was observed at 48 hr. Cell cycle kinetic parameters were correlated with the appearance of cell mediated cytotoxicity.

Animals↗
PubMed2003

Multicenter quality control for the detection of hepatitis C virus RNA in seminal plasma specimens.

The discrepant results available in the literature about the presence of hepatitis C virus (HCV) RNA in seminal plasma of men chronically infected by this agent are related, at least in part, to the molecular techniques used and particularly to the wide range of protocols dedicated to RNA extraction. In order to evaluate these protocols and to standardize the method of detection of HCV RNA in this fluid, a panel of coded specimens was tested blindly in 12 French laboratories; it included 14 seminal plasma specimens and four water controls spiked with HCV RNA ranging from 10 to 20000 IU/ml and two HCV-negative seminal plasma specimens. The extraction step was performed according to methods using either silica beads (NucliSens [Organon Teknika S.A., Fresnes, France]; RNA viral kit [Qiagen, Courtaboeuf, France]) or guanidinium thiocyanate (Amplicor HCV assay; Roche Diagnostics, Meylan, France), preceded or not by a centrifugation of the seminal plasma. For the amplification step, all the laboratories performed the same reverse transcription-PCR technique (Amplicor HCV Cobas assay). The percentage of correct results ranged from 53.3 to 100, the poorest results being obtained when no centrifugation step preceded the Amplicor extraction protocol. The rate of correct results was significantly higher in laboratories using a preliminary centrifugation of the specimen (P = 0.034 by chi-square test). By contrast, the overall number of correct results was not correlated to the initial volume of sample used for the test. These results allowed us to validate standardized techniques adapted to the performance of this test on a routine basis, especially in men infected with HCV and involved in programs of medically assisted reproduction.

Hepacivirus↗
PubMed1960

Biological properties of ribonucleic acid from virulent and attenuated poliovirus.

Concentrates of type I poliovirus were extracted with phenol by Gierer and Schramm's method. The infectivity of these extracts in tissue culture and mice appeared to be associated with viral RNA. Studies on neurovirulence based on the "t" marker and intraspinal pathogenicity in mice demonstrated that the viral progeny of RNA isolated from virulent viruses had the "t(+)" character and were highly neurotropic for mice. Conversely, RNA extracted from attenuated virus gave rise to a progeny of "t" type which were avirulent for mice. The results suggest that the infective RNA from type, I poliovirus carries the genetic information which determines the degree of neurovirulence.

Animals↗
PubMed1992

Detection of laminin receptor mRNA in human cancer cell lines and colorectal tissues by in situ hybridization.

The 67-kd high-affinity laminin receptor (67 LR) is a gene product whose expression appears to be associated with the invasive and metastatic phenotype of a variety of human cancer cells. Northern blot hybridization has been routinely used to quantify the level of 67 LR mRNA from total cellular RNA extracts of homogenized tissue specimens or in vitro grown cell populations. This technique is useful to assess the average expression of the 67 LR mRNA of a particular sample but does not provide information about expression in specific cell types nor about heterogeneity of expression from cell to cell. In this study, we analyzed the expression of 67 LR mRNA in four human cancer cell lines with varying degrees of expression of 67 LR protein (renal cancer A-704, breast carcinoma MCF-7/4 and MCF-7/7, and pancreatic cancer Panc-1) using in situ hybridization performed with 67 LR riboprobes. Total cellular RNA was simultaneously extracted from the cell lines and hybridized on Northern blots with a 67 LR cDNA probe to assess the validity of the mRNA detection by in situ hybridization. Sixty-seven LR mRNA expression was higher in Panc-1 and MCF-7/4 cells than in MCF-7/7 and renal carcinoma A-704. There was a direct correlation (R2 = 0.88) between the in situ hybridization analysis and the mRNA levels detected by Northern blot analysis. The in situ hybridization method showed a heterogeneous expression of the 67 LR mRNA in the four cell lines with different subpopulations of cells showing a range from negative to high levels of the message. Sixteen freshly frozen human colorectal tissues (seven adenocarcinomas, five matched normal mucosae, and four adenomas) were also analyzed by in situ hybridization. The 67 LR mRNA was localized in normal and neoplastic epithelial cells. Adenocarcinoma cells showed a 1.6- to 5-fold higher expression (P < 0.02 according to the Wilcoxon-Mann-Whitney test) than did epithelial colonic cells from normal mucosae or adenomas. The signal tended to be stronger in poorly differentiated carcinomas and carcinomas with metastases than in moderately differentiated and nonmetastatic tumors. We conclude that the high expression of 67 LR mRNA in colorectal tumors is due to an increased production by tumor cells. Furthermore, in situ hybridization is an effective method to detect the expression of LR mRNA in cultured cell lines as well as in frozen tissue sections.

Adenoma↗
PubMed1993

Early strain-related changes in cultured embryonic chick tibiotarsi parallel those associated with adaptive modeling in vivo.

A model was developed for the application of cyclic mechanical loads to 17 day embryonic chick tibiotarsi in culture. A single 20 minute period of intermittent loading at 0.4 Hz, producing physiologic peak strains and strain rates, resulted in two peak strain magnitude-related responses that were previously reported in vivo: (1) a rapid increase in glucose 6-phosphate dehydrogenase activity in osteoblasts and osteocytes and (2) increased RNA synthesis, as shown by increased incorporation of [3H]uridine into extracted RNA. The RNA response was detectable 8 h following loading but was more pronounced by 24 h. Both responses were blocked by indomethacin (10(-6) M). These results demonstrate that embryonic chick bones in organ culture exhibit cellular responses to loading similar to those previously identified in adult canine cancellous bone cultures in vitro and adult avian cortical bone in vivo. These findings are consistent with a sequence of events between loading and new bone formation that includes an immediate strain magnitude-related, prostanoid-dependent increase in activity of the pentose monophosphate shunt in osteoblasts and osteocytes, followed by a similarly strain magnitude-related increase in RNA synthesis over the subsequent 24 h.

Adaptation, Physiological↗
PubMed2003

Differential gene expression analysis using paraffin-embedded tissues after laser microdissection.

Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.

Acetic Acid↗
PubMed1985

Methylation of 16S ribosomal RNA and resistance to the aminoglycoside antibiotics gentamicin and kanamycin determined by DNA from the gentamicin-producer, Micromonospora purpurea.

When DNA fragments from Micromonospora purpurea (the producer of gentamicin) were cloned in Streptomyces lividans, a gentamicin-resistant strain was obtained in which the ribosomes were highly resistant both to gentamicin and to kanamycin. Reconstitution analysis revealed that such resistance resulted from some property of their 16S RNA. Extracts from the clone contained methylase activity which acted on 16S RNA within E. coli 30S ribosomal subunits and rendered them resistant to gentamicin and kanamycin.

DNA, Bacterial↗
PubMed1986

Accumulation and specific cleavage of 5S RNA in the isthmus of laying hen oviduct. Evidence for three chicken 5S RNA.

RNA extracts from the isthmus of laying hen oviduct contain truncated 5S RNA molecules that were found to be shorter at their 5' terminus as compared to native 5S RNA I and II. Moreover one of the truncated species differs from 5S RNA I by the absence of the 3' end nucleotide. The truncated forms increase of about 70% the total 5S RNA (intact + truncated) in the isthmus, as compared to the other studied tissues. Furthermore 5S RNA I is heterogeneous: 25% have A instead of U at the 3' end, and some evidence was obtained for the existence of two 5S RNA I conformers.

Animals↗
PubMed1978

Anti-tumour cytotoxicity of poly(A)-containing messenger RNA isolated from tumour-specific immunogenic RNA.

The transfer of tumour-specific cytotoxicity against a murine fibrosar-coma has been demonstrated in vitro using xenogeneic RNA extracted from tumour-cell-immune animals. Poly(A)-tailed messenger RNA from immunogenic RNA was isolated by passage through an oligo(dT)-cellulose column, and evaluated to determine whether the same tumour-specific cytotoxicity could be transferred. Aliquots of normal C3H mouse lymphocytes were treated with poly(A)-containing immune RNA, whole-cell immune RNA lacking poly(A) and total cellular immune RNA. Treated cells were tested in vitro using an adaptation of the Takasugi and Klein microcytotoxicity assay. Percent cytotoxicity was calculted using cells treated with fractions of normal RNA as control. An increase in tumour cytotoxicity was found with poly(A)-containing immune RNA. The optimum dose of poly(A)-tailed immune RNA was estimated as 6.5 microgram of RNA per 4 x 10(6) lymphocytes. Populations of lymphocytes were separated using glass and nylon wool. T- and B-enriched populations were treated with various RNA components. The adherent cell population showed no significant cytotoxicity, whilst treatment of the nonadherent population with poly(A)-tailed immune RNA produced high levels of cytotoxicity.

Animals↗
PubMed2001

Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors.

The expression of neurotrophins and neurotrophin receptors in non-neural tissue is related to tissue remodeling, differentiation, proliferation and migration of target cells. The literature yields contradictory results on neurotrophin and neurotrophin receptor expression in the liver. We show immunoreactivity to antibodies to nerve growth factor (NGF), brain-derived neurotrophin (BDNF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5), the low-affinity nerve growth factor receptor p75 and the high-affinity tyrosine kinase receptors (Trk) B and C in hepatic stellate cells and weak reactivity for BDNF, NT-3, and NT-4/5 in hepatocytes, in cryosections of human and rat liver, in normal and varying pathologic conditions. Immunoreactivity is unequivocally localized to hepatic stellate cells by double staining with alpha-smooth muscle actin (alpha-SMA) and desmin, studied by confocal laser scanning microscopy. Finally, the presence of mRNA transcripts for the different neurotrophins and neurotrophin receptors, with the exception of Trk-B, is shown by reverse transcription polymerase chain reaction (RT-PCR) on RNA extracted from freshly isolated rat hepatic stellate cells, compared with hepatocyte RNA. Hepatocyte RNA was found to contain BDNF, NT-3, NT-4/5 mRNA (which is compatible with the immunohistochemical findings) and Trk-A mRNA. In conclusion, hepatic stellate cells are a source of several neurotrophins in the liver and they express neurotrophin receptors. These findings correspond with the known involvement of hepatic stellate cells in tissue remodeling, their production of extracellular matrix components and their proliferation in acute necrotizing liver pathology. In analogy with findings in other organs and systems, neurotrophins are hypothesized to play a role in the pathophysiology of liver disease.

Animals↗