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 1,153 records · Page 64Linked to original sources
PubMed1968

Separation of ten reovirus genome segments by polyacrylamide gel electrophoresis.

Double-stranded ribonucleic acid (RNA) extracted from purified reoviruses of all three serotypes and from type 3 virus-infected cells was analyzed by polyacrylamide gel electrophoresis. It was calculated that each RNA includes 10 segments: 3 large, 3 intermediate, and 4 small fragments corresponding to molecular weights of about 2.5, 1.4, and 0.8 x 10(6) daltons, respectively, or a total of 15 x 10(6) daltons.

Acrylates↗
PubMedDate not supplied

[Characterisation of RNA from synchronously growing yeast cells (Saccharomyces cerevisiae) as separated by columns of methylated albumin on Kieselgur (MAK) (author's transl)].

Total RNA, extracted from synchronously growing yeast cells by the method of Georgiev-Mantieva, was separated by columns of methylated albumin on Kieselgur (MAK). By comparison with definited RNA's in discelectrophorese and after pulse labelling with [3H]methionine the following fractions are identified: t-RNA, 5.8S RNA, m-RNA, 18S and 25S R-RNA and their precursors 20S, 27S and 35S. High molecular fractions of RNA between 40S and 70S were also found.

Chromatography↗
PubMed1984

Limitation of differential expression of HLA-A,B,C antigens on choriocarcinoma cell lines by messenger RNA for HLA heavy chain but not by beta 2-microglobulin.

The relative amounts of HLA-A,B,C antigens, beta 2-microglobulin (beta 2m), and trophoblast antigens (Trop-1 and Trop-2) were determined on nine choriocarcinoma cell lines including seven lines of gestational origin and two lines of nongestational origin (from ovary and stomach) by quantitative immunofluorescence analysis using a fluorescence-activated cell sorter. Most of these lines expressed surface HLA to variable extents, but one had none detectable. However, all lines secreted readily measurable amounts of beta 2m. We analyzed total RNA extracted from these lines using northern blot molecular hybridization with HLA-A,B,C- and beta 2m-specific complementary DNA probes. We found no messenger RNA species which hybridized with the HLA probe in cells with no detectable HLA surface antigen and only small amounts of HLA-specific RNA in cells with low levels of HLA membrane antigen. Cells exhibiting surface HLA levels greater than about 30% of that on lymphocytes had much higher amounts of HLA-specific RNA than did choriocarcinoma cells with no or low HLA antigen expression. In contrast, RNA hybridizing with beta 2m-specific probes was present at the 20% level or higher (relative to lymphocytes) in all the cell lines tested. Thus, the expression of HLA-A,B,C is apparently limited in choriocarcinoma cells by the level of HLA heavy-chain RNA and not by the level of beta 2m RNA. We discuss these findings in relation to the normal trophoblastic or other origins of this tumor type and with respect to the regulation and function of HLA in trophoblasts.

Antibodies, Monoclonal↗
PubMed1995

In situ amplification of measles virus RNA by the self-sustained sequence replication reaction.

BACKGROUND: The self-sustained sequence replication (3SR) reaction is an isothermal method for nucleic acid amplification that has several features that make it an attractive alternative to PCR. We have studied the feasibility of the in situ 3SR reaction in cells using a measles virus-infected cell line as a model. EXPERIMENTAL DESIGN: The study was carried out in four steps. First, using RNA extracted from a measles-infected Vero Green monkey kidney cell line, conditions for the in vitro amplification of a segment of the nucleocapsid portion of the RNA viral genome were optimized for 420- and 119-bp 3SR products, and the results were compared. Second, 3SR was performed on intact infected cells in suspension, and the amount of RNA product was compared with infected cells without 3SR. Then, the 3SR reaction was conducted on cytospin preparation slides, followed by in situ hybridization for detection of the amplification product. Finally, 3SR was carried out on sections of formalin-fixed, paraffin-fixed, paraffin-embedded cells, and the degree of amplification as detected by ISH was quantified and compared between infected cells with and without 3SR reaction. RESULTS: Specific amplification of measles was observed in each of these types of preparations with an 8.5-fold rate of amplification in paraffin sections of formalin-fixed cells (a mean of 272.5 +/- 65.3 grains/cell after 3SR amplification in comparison to 31.97 +/- 4.2 grains/cell without amplification). CONCLUSIONS: A significant amount of amplification of RNA is possible with in situ 3SR (IS-3SR) and, in combination with ISH, offers several advantages compared with in situ PCR (IS-PCR), such as ease of use, lack of conditions that lead to cell damage, and a specificity for RNA amplification. This is the first report of specific amplification of RNA within cells using the IS-3SR procedure, a technique that has a wide range of potential applications in pathology and molecular biology.

Animals↗
PubMed1998

Transferrin production by the ciliary body of rabbits: a biochemical and immunocytochemical study.

PURPOSE: We have previously reported that transferrin is one of several glycoproteins synthesized within the eye and secreted into the vitreous. The present investigation was designed to determine the role of the ciliary body in the production of this vitreous transferrin. METHODS: Isolated ciliary body-iris were incubated with 3H-fucose, 3H-tyrosine or 35S-methionine and afterwards the culture media were processed for affinity chromatography using columns of Sepharose conjugated with antibody to rabbit plasma transferrin. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using total RNA extracted from fresh ciliary body-iris and primers constructed on the basis of the known sequence of transferrin mRNA from rabbit liver. The fragment obtained was employed as a probe in northern-blots of total RNA of ciliary body-iris. Furthermore, paraffin sections of eyes were treated for immunocytochemical visualization of transferrin. RESULTS: A labeled polypeptide, specifically eluted from the antitransferrin columns, was detected in the incubation medium, transferrin mRNA was found in extracts of whole ciliary body-iris, and transferrin antigenicity was identified in the ciliary and iridial epithelial cells by immunocytochemistry. CONCLUSIONS: These results demonstrate the ciliary epithelium as one of the sources of the vitreous transferrin.

Animals↗
PubMed2003

Sheep heart RNA stimulates myofibril formation and beating in cardiac mutant axolotl hearts in organ culture.

In the Mexican axolotl, Ambystoma mexicanum, recessive mutant gene c, when homozygous, results in a failure of the heart to form sarcomeric myofibrils and contract normally. Previous studies have shown that purified RNA from normal anterior endoderm or from medium conditioned with anterior endoderm/pre-cardiac mesoderm has the capacity to rescue mutant hearts in organ culture. In the present study, RNA extracted from adult sheep heart was tested for its capacity to promote differentiation in the mutant axolotl hearts. Mutant hearts cultured in the presence of the sheep heart RNA in Steinberg's solution for 48 h displayed rhythmic contractions. Ultrastructural studies showed that the rescued mutant axolotl ventricular myocardial cells contained myofibrils of normal morphology. Mutant hearts cultured in Steinberg's solution alone did not beat throughout their lengths and myofibrils were not observable in the ventricles. Confocal microscopy confirmed the increase of Tropomyosin expression and formation of myofibrils in mutant hearts treated by sheep heart RNA. Thus, sheep heart RNA promotes myofibrillogenesis and the development of contractile function in embryonic cardiac mutant axolotl hearts.

Ambystoma mexicanum↗
PubMed1989

Isolation and analysis of ribonucleic acids from skeletal tissues.

We report a method for the isolation of total cellular RNA from mineralized or cartilaginous tissues. The procedure accommodates the large amount of hydroxyapatite and high buoyant density proteoglycans present in skeletal tissue samples, as well as the low cell density characteristic of these tissues. The procedure can be reliably used for processing a large number of small (100-800 mg) tissue samples. Tissues are homogenized in guanidine hydrochloride solution, then centrifuged at low speed, and filtered to remove the nonsolubilized extracellular matrix proteins. Subsequent high speed density gradient centrifugation produces a high yield of RNA (0.2-0.6 micrograms RNA/mg tissue) which is precipitated in a low pH sodium acetate solution. RNA extracted by this method has been analyzed for the expression of various genes by Northern blotting. In addition to mRNAs of bone- and cartilage-specific proteins, messenger RNA for growth factors, proto-oncogenes, and heat shock proteins can be detected.

Animals↗
PubMed1984

Genome subunit reassortment among Bunyaviruses analysed by dot hybridization using molecularly cloned complementary DNA probes.

A simple and rapid procedure for determining the genotypes of viruses has been applied to analysis of genome subunit reassortment in heterologous crosses of Batai virus, Bunyamwera virus, and Maguari virus, three members of the Bunyamwera serogroup of bunyaviruses. The procedure for determining genotype made use of specific molecular probes to identify the parental origin of the L and M RNA subunits. Complementary DNA copies of the three RNA segments of Bunyamwera virus were prepared by reverse transcription using synthetic oligonucleotide primers for first and second strand synthesis. The cDNA transcripts were inserted into a pBR322 vector and gene-specific probes prepared from nick-translated plasmid DNA. L and M gene-specific probes were identified which could unequivocally discriminate Bunyamwera virus genome subunits in a dot-hybridization test using cytoplasmic RNA extracts immobilised on nitrocellulose filters. None of the S gene-specific probes were sufficiently discriminatory for use in this test. Instead the parental origin of the S RNA subunit was inferred from the electrophoretic mobility of the virion N protein. It was observed that reassortment did not occur at random in heterologous crosses of is mutants of the three viruses, and only the M RNA subunit appeared to segregate freely. However, unrestricted reassortment was observed when recombinant viruses with nonhomologous subunit combinations were used as the parental viruses. It was concluded, therefore, that restriction was mediated at the gene product level and that nonrandom reassortment was not due to incompatibility of genome subunits.

Animals↗
PubMed1979

In vitro synthesis of a 5S RNA precursor by isolated nuclei of rat liver and HeLa cells.

Isolated rat liver nuclei were incubated under appropriate conditions in the presence of 0.5 micrograms/ml alpha-amanitin and an RNAase inhibitor prepared from cytosol fraction, together with alpha-32P-UTP or alpha-32P-CTP and three other nucleoside triphosphates. RNA extracted by an SDS-hot phenol procedure was fractionated with sucrose density gradient centrifugation followed by acrylamide gel electrophoresis. Fingerprint analysis of the in vitro synthesized "5S" RNA, which was slightly larger than mature 5S RNA on gel electrophoresis, showed that it contained all the sequences of mature 5S RNA except for the oligonucleotide at the 3' end. Instead, it contained two additional spots which were not present in mature 5S RNA. Analysis of the extra spots revealed that they were derived from the 3' end of the in vitro synthesized "5S RNA, which were sequenced tentatively as -CUUGAUGCUUoh (extra sequence underlined). The 5' end of the product was (p)pGU--. Isolated HeLa cell nuclei synthesized similar sized "5S" RNA under the same conditions. We conclude from these results that in isolated nuclei of these mammalian cells RNA polymerase III starts transcription of 5S RNA gene at the same site as the 5' end of mature 5S RNA, proceeds toward the 3' direction and stops at a site probably 8 nucleotides downstream from the 3' end of mature 5S RNA. Experiments with a short pulse and with various "chases" have demonstrated the presence of a short-lived precursor 5S RNA which is similar in size and sequence to in vitro "5S" RNA, suggesting that 5S RNA is synthesized in vivo as a longer precursor molecular as demonstrated in this in vitro system, and is rapidly processed into mature 5S RNA.

Animals↗
PubMed1991

Insulin-like growth factor-I gene expression during development and estrous cycle in the rat uterus.

The ontogeny and estrous cycle-dependent variation of insulin-like growth factor-I (IGF-I) gene expression was analyzed in the rat uterus. RNA extracted from rat uteri contained transcripts with estimated sizes of 7.0, 1.7, and 1.2-0.8 kb that were recognized by a 32P-labelled mouse IGF-I RNA probe. A solution hybridization RNase protection assay was used to measure the abundance of IGF-I mRNAs in uteri from rats of different ages. The highest levels were found in adult rats (p less than 0.01). The levels of IGF-I transcripts changed markedly during the estrous cycle with the highest levels at proestrus (p less than 0.01). There was an 8-fold increase in the abundance of IGF-I mRNA between diestrus-2 and proestrus. The corresponding livers had no significant variation of IGF-I gene expression during the estrous cycle, demonstrating a tissue-specific regulation of the IGF-I gene. The time and dose dependency of estrogen regulation of IGF-I gene expression was studied in hypophysectomized rats. The levels of IGF-I mRNA in the uterus decreased after hypophysectomy. A single s.c. injection of estradiol significantly increased the levels of IGF-I transcripts after 3 h (p less than 0.01). A low dose of estradiol (0.1 micrograms/100 g) increased the levels of IGF-I transcripts but progesterone in higher doses (5 micrograms/100 g) was without effect, indicating that the effect was specific for estradiol. However, the present study provides no information regarding whether this regulation is at the level of transcription or mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1990

Regulation of sucrase-isomaltase gene expression along the crypt-villus axis of rat small intestine.

The expression of sucrase-isomaltase mRNA was investigated along the crypt-villus axis of rat small intestine using differentially isolated cells and in situ hybridization. A partial rat sucrase-isomaltase cDNA was cloned which coded for a protein that was predicted to be 88% homologous to those encoded by the rabbit and human cDNAs. Southern blot analysis of rat genomic DNA indicated that the cDNA hybridized to a single gene. Northern blots of RNA extracted from subpopulations of intestinal epithelial cells that were isolated from villus and crypt compartments showed that this cDNA hybridized to a 6.5 kb band predominantly in villus RNA. In situ hybridization using 35[S]-labeled RNA probes demonstrated that autoradiographic grains were detected over eptithelial cells located on villi with the greatest number of grains located at the crypt-villus junction and in the lower to mid-villus region; from mid-villus to the villus tip there was a decline in sucrase-isomaltase mRNA. We conclude that expression of sucrase-isomaltase as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation which, most likely, is a result of activation of sucrase-isomaltase gene transcription.

Amino Acid Sequence↗
PubMed2002

Efficient isolation of total RNA from antibiotic-producing bacterium Amycolatopsis mediterranei.

RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA. Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality. The genus of Amycolatopsis mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus. In this report, we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis. Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA. About 1 microg total RNA free of DNA contamination can be obtained from 1 mg wet weight of A. mediterranei. The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses.

Actinomycetales↗
PubMed1967

Synthesis of ribonucleic acid in normal bone in vitro.

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.

Animals↗
PubMed1984

Myosin heavy chain mRNA during the development and regression of myocardial hypertrophy.

RNA synthesis increases during the development and decreases during the regression of pressure-overload induced myocardial hypertrophy. Until now, we have been unable to determine whether these events actually reflected changes in the messenger RNAs for the myosin heavy chain mRNA in the total RNA extracted from rat heart during the development and regression of hypertrophy. We found that the amount of MHC mRNA increased in proportion to the overall increase in total RNA during the development of hypertrophy. However, upon relief of pressure-overload, MHC mRNA decreased to a much greater degree than did total RNA. This is probably related to a sudden cessation of synthesis and the rapid degradation of mRNA. We conclude that the development and regression of myocardial hypertrophy is a function of changes in the mRNA for contractile proteins.

Animals↗
PubMed1976

Polyadenylate sequences of human rhinovirus and poliovirus RNA and cordycepin sensitivity of virus replication.

The polyadenylate [poly(A)] content of the genome RNA of human rhinovirus type 14 (HRV-14) is nearly twice as large as that of the genome RNA of poliovirus type 2. The poly(A) content of viral RNA was determined to be the RNase-resistant fraction of 32P-labeled viral RNA extracted from purified virions. Polyacrylamide gel electrophoresis indicated that the poly(A) sequences of HRV-14 are more heterogenous and on an average larger than those of poliovirus RNA. On the basis of susceptibility to micrococcal polynucleotide phosphorylase the rhinovirus genome terminates in poly(A). Replication of both viruses is almost totally inhibited by cordycepin at 50 mug/ml. At lower concentrations, rhinovirus replication is more sensitive to cordycepin than poliovirus replication. Addition of cordycepin (75 mug/ml) to infected culture prior to or during viral RNA replication results in more or less complete inhibition of virus-specific RNA synthesis. The results do not indicate that cordycepin sensitivity of either virus is due to preferential inhibition of viral poly(A) synthesis by this antibiotic.

Base Sequence↗
PubMed1999

[Effects of methyl tertiary butyl ether on liver tissue gene expression in rats].

OBJECTIVE: Methyl tertiary butyl ether (MTBE) is a new gasoline additive, with certain carcinogenecity in animal experiments. To study its possible mechanism of carcinogenesis in animals, expression of protooncogene c-myc and functional gene glutathione S-transferase-P (GST-P) in the liver tissues of rats chronically exposed to MTBE was detected. METHODS: Forty male SD rats with body weights of 180 - 200 grams were randomly divided into four groups and gavaged with MTBE dissolved in soybean oil with doses of 1 000, 600, 200 and 0 mg/kg, respectively, once daily and five days a week for 13 weeks. Liver tissues of the animals were frozen quickly in liquid nitrogen and their total RNA extracted with one-step method. The probes of c-myc and GST-P were labeled with digoxin by random primer method, and dots hybridization with RNA was used. RESULTS: Levels of expression of c-myc gene in the liver tissues of rats increased significantly, but not for those of GST-P. CONCLUSION: MTBE can induce the higher expression of c-myc gene, which suggests it can promote cell proliferation-one of possible mechanisms of carcinogenesis in animals.

Animals↗
PubMed1996

[Levels of cytokeratin CK19 expression in mononuclear blood cells evaluated using a reverse PCR (RT-PCR)].

AIMS: The sensitivity and specificity of a reverse transcription PCR method (RT-PCR) to detect cytokeratin K19 (CK19) expression was evaluated with the purpose of assessing its capability to detect the presence of breast cancer tumour cells in peripheral blood progenitor cell collection that had to be reinfused to breast cancer patients submitted to intensive chemotherapy as haematopoietic support. PATIENTS AND METHODS: Two breast cancers as positive samples were used and 34 samples of mononucleated blood cells as negative controls: 18 peripheral blood samples from normal subjects, 14 from different types of leukaemias (M3, M4Eo, M2, etc.) and two from two patients with Hodgkin's lymphoma. The method studied is a nested RT-PCR that amplifies the CK19 expression from the sample RNA extracted following the method of phenol-chloroform. RESULTS: The right performance of the method is demonstrated by observing the detection of CK19 transcripts in the breast cancer RNA and by obtaining good blank results both with non transcribed RNA and with DNA. Moreover, the method has an excellent sensitivity such as to allow the detection of CK19 transcripts in a 10(-6) dilution of cDNA reverse transcribed from 1 microgram of breast cancer RNA. The CK19 transcripts were also detected in the 64% of RNA obtained from the mononucleated blood cells controls, although the percentage of positivities was lower (47%) in the RNA from peripheral blood samples. Nevertheless it should be remarked that the levels of CK19 expression in the blood mononucleated cells is almost negligible since it used to extinguish at 1:5 cDNA dilution. CONCLUSIONS: The method studied is specific and has a high sensitivity that explains the detection of CK19 illegitimate expression approximately a half in mononucleated blood cells negative controls. However, the levels of CK19 expression in mononucleated blood cells were almost negligible and it used to extinguish at 1:5 cDNA dilution, therefore it could be concluded that the method might be useful to detect breast cancer occult tumours cells in mononucleated blood cell collection, always provided that a lower amount of cDNA is taken, thus decreasing to nil almost the false positive samples and keeping always a good sensitivity.

Biomarkers, Tumor↗
PubMed1981

Coronavirus JHM: characterization of intracellular viral RNA.

After infection of Sac(-) cells with the murine coronavirus JHM the synthesis of seven major and two minor RNA species was induced. These RNAs were polyadenylated and single-stranded. Their mol. wt. were estimated by electrophoresis in agarose gels containing methylmercury hydroxide. The values for the major species were 6.67 x 10(6) for RNA of genome size (RNA 1), 3.42 x 10(6) for RNA 2, 2.76 x 10(6) for RNA 3, 1.35 x 10(6) for RNA 4, 1.19 x 10(6) for RNA 5, 0.93 x 10(6) for RNA 6 and 0.62 x 10(6) for RNA 7. The minor species have a size of 4.7 x 10(6) (RNA a) and 1.5 x 10(6) (RNA b). The same number of species were found by electrophoresis after denaturation with glyoxal-dimethyl sulphoxide. No gross difference in number of RNAs and the amount of each species was found between total cytoplasmic RNA, polyadenylated cytoplasmic RNA and RNA extracted from pelleted polysomes.

Coronaviridae↗