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PubMed2004

Utility of linearly amplified RNA for RT-PCR detection of chromosomal translocations: validation using the t(2;5)(p23;q35) NPM-ALK chromosomal translocation.

The requirement for sufficient quantities of starting RNA has limited the ability to evaluate multiple transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR). In this study, we demonstrate the utility of linear RNA amplification for RT-PCR analysis of multiple gene transcripts including a chromosomal translocation, using the t(2;5)(p23;q35) as a model. RNA from the t(2;5)-positive cell line, SU-DHL-1, and the t(2;5)-negative cell line, HUT-78, was extracted and exposed to two rounds of linear amplification. RT-PCR using cDNA from the resultant amplified (a) RNA and total RNA resulted in the 177 bp NPM-ALK fusion gene product from the SU-DHL-1 cell line, but not from aRNA or total RNA from the HUT-78 cell line. DNA sequencing of the RT-PCR products from total and aRNA of SU-DHL-1 cells demonstrated identical sequences corresponding to the NPM-ALK fusion gene. Evaluation of 25 snap-frozen tissue samples, including eight NPM-ALK-positive ALCLs demonstrated 100% concordance of t(2;5) detection between cDNA from total RNA and that from aRNA. Our results show that linear amplification of RNA can enhance starting RNA greater than 200-fold and can be used for rapid and specific detection of multiplex gene expression from a variety of sources. This method can generate a renewable archive of representative cDNA, which can be used for retrospective screening of stored samples as well as positive controls for the clinical molecular diagnostic laboratory.

Cell Line, Tumor↗

PubMed1988

Pregnancy-specific beta 1 glycoprotein in rat: tissue distribution of the mRNA and identification of testicular cDNA clones.

A partial cDNA of pregnancy-specific beta 1 glycoprotein isolated from human term placenta was used as probe for slot-blot analysis of total RNA extracted from placental and non-placental tissues in the rat. RNA hybridization with the probe was observed in rat placenta, indicating the presence of mRNA highly homologous to human SP1. The quantity of hybridizing RNA increased with increasing gestational age. In non-pregnant rats, SP1-hybridizing mRNAs were found in uterus, intestine and testis, while no hybridizing material was detected in liver or muscle. The amount of rat SP1 mRNA, based on percentage of total tissue RNA, was greatest in the testis followed by intestine, uterus and placenta. Using the same probe, six clones were obtained by screening a rat testis cDNA library. These clones carried cDNA inserts ranging in size from 1530 to 1983 bp. An internal EcoRI site was present in all cDNA clones. Southern blot analysis confirmed that the cDNA insert of all the clones was homologous to human placental SP1 cDNA. These results suggest a possible origin for the trace quantities of SP1 detected in non-pregnant individuals. It also confirms that the rat is an appropriate model for studying the physiological functions of SP1.

Animals↗

PubMed1989

Preparation of RNA from unspheroplasted yeast cells (Saccharomyces cerevisiae).

High-quality RNA can be prepared from up to 100-ml culture volumes of unspheroplasted yeast cells (Saccharomyces cerevisiae) via homogenization in high-temperature phenol:chloroform mixtures. The yield of RNA from this preparative method is equivalent to those of other methods requiring preliminary spheroplasting of cells. Quality and quantity of recovered RNA are independent of yeast strain and cell growth medium used, and the method works equally well on cells in either log phase growth or in stationary phase. Mitochondrial RNAs recovered as part of whole cell RNA mixtures may be slightly degraded. Analyses of individual transcripts in the recovered RNA mixtures suggest that there is no selection for or against any specific single transcript or any group of transcripts when RNA is prepared by this method.

Blotting, Northern↗

PubMed1986

Lack of correlation between the accumulation of plus-strand leader RNA and the inhibition of protein and RNA synthesis in vesicular stomatitis virus infected mouse L cells.

The inhibition of protein synthesis in mouse L cells infected by vesicular stomatitis virus (VSV) requires expression of two regions (one large and one small) of the viral genome, as determined by target size analysis. The inhibition of host RNA synthesis was also shown to be dependent on expression of two regions of the VSV genome, most likely the same ones. In some cases, such as in cells infected by mutants T1026R1, or tsG41 at 40 degrees, or moderately uv irradiated VSV, only one of the two regions was expressed, yet cellular protein and RNA synthesis was decreased. This suggests that the product of each region of the viral genome can act independently. In these instances the severity of the inhibition was dependent on both the length of the infection period and the multiplicity of infection. The identity of neither gene product is known, but it has been suggested that small product is plus-strand leader RNA. As shown herein, however, there was no correlation between the extent of host macromolecular synthesis inhibition and the quantity of leader RNA in infected cells.

Animals↗

PubMed1975

Translation and identification of the mRNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

Vesicular stomatitis virus messenger RNA has been transcribed in vitro from the viral genome by the virion-associated RNA polymerase in quantities suitable for translation. Wheat germ cell-free extracts programmed with the isolated in vitro 12-18S RNA fraction synthesize polypeptides similar to the viral N, NS, and M proteins, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping of the in vitro products and the viral marker polypeptides. In addition, the RNA synthesized in vitro also codes for a protein of molecular weight 63,000 which may be a nonglycosylated form of the viral glycoprotein G. The 12-18S RNA has been partially separated into individual messenger species and these have been identified by the proteins for which they code. There are four monocistronic messenger species in the in vitro 12-18S RNA and the coding capacity of three of these molecules agrees with the estimated molecular weight of the polypeptide assigned to it.

Animals↗

PubMed1989

Regulation of microtubule-associated protein 2 (MAP2) mRNA expression during rat brain development.

The expression of MAP2 during rat brain development was studied by using specific antibodies and cDNA probes. MAP2 cDNAs were isolated from a rat brain lambda gt11 library, and their identity was confirmed by the reactivity of their fusion proteins with several independent monoclonal antibodies that recognize MAP2. Northern blot analyses of the RNA prepared from whole brains, cerebral cortex, hypothalamus, brain stem, olfactory bulbs, and cerebellum showed that the levels of MAP2 mRNA increase during the initial phase of development, reach a maximum between postnatal weeks 2 and 3, and then decrease in the adult. The time course and the kinetics of this change varied between different brain regions and appeared to reflect the pattern of morphological changes in these regions. RNA blots were also analyzed with beta-tubulin and beta-actin cDNA probes to ensure the quality and the quantity of the RNA. The levels of MAP2 mRNA and protein showed similar changes during the initial part of brain development and suggested a transcriptional control. However, while MAP2 protein levels remained high throughout development, MAP2 mRNA levels decreased in adulthood. We suggest that the increased stability of the MAP2 molecule may be a contributing factor in the developmental regulation of steady-state levels of MAP2.

Actins↗

PubMed1966

Effect of cycloheximide on protein and ribonucleic acid synthesis in cultured human lymphocytes.

1. Phytohaemagglutinin stimulates the transformation into blast cells of human lymphocytes incubated in vitro. This transformation is accompanied by an increase in the incorporation of [(14)C]leucine into protein and [(3)H]uridine into RNA. 2. The incorporation of [(14)C]leucine by cultures grown in the presence or absence of phytohaemagglutinin is inhibited to the same extent by cycloheximide, a known inhibitor of protein synthesis. 3. Lymphocytes grown without phytohaemagglutin synthesize mainly non-ribosomal RNA. [(3)H]Uridine incorporation by these cells was increased by cycloheximide. 4. Lymphocytes incubated with phytohaemagglutinin begin to synthesize substantial quantities of ribosomal RNA. Under these conditions [(3)H]uridine incorporation was partially inhibited by cycloheximide. This inhibition is shown to be largely a result of inhibition of the synthesis of ribosomal RNA.

Amino Acids↗

PubMed2003

Rapid extraction of RNA and analysis of transcript levels in Chlamydomonas reinhardtii using real-time RT-PCR: Magnesium chelatase chlH, chlD and chlI gene expression.

The reverse transcriptase polymerase chain reaction (RT-PCR) is an extremely sensitive technique for detecting RNA transcripts. Real-time RT-PCR using fluorescent dyes and instruments such as the Roche Lightcycler allows real-time kinetic quantification of transcript levels. Here we report a method for the relative quantification of RNA transcripts using real-time RT-PCR that gives results comparable to Northern blotting utilizing 10-100 fold less RNA. We have also optimized a method for the rapid and efficient extraction of RNA from Chlamydomonas reinhardtii. The method is more rapid than other methods tested, allows simultaneous processing of multiple samples, and yields reproducible quantities of total RNA from a fixed number of cells. In addition the purified total RNA is of high quality and polyA-mRNA can be easily isolated. Using these methods we found that the pattern of changes in RNA transcript levels of the magnesium chelatase (Mg-chelatase) genes, chlH, chlD and chlI, of C. reinhardtii grown under synchronous culture conditions in light/dark cycles are similar and that light is involved in this regulation.

Journal Article↗

PubMed1995

High hepatitis C viraemia and impaired antibody response in patients coinfected with HIV.

OBJECTIVE: To compare hepatitis C virus (HCV) load in patients infected with HCV alone and those coinfected with HIV, and to evaluate the antibody response to HCV in the case of HIV infection. DESIGN: Patients coinfected with both HCV and HIV have been shown to develop hepatic changes more rapidly, which may be due to an interaction between HCV and HIV. In a prospective study, serum samples were taken from 150 patients. METHODS: Using reverse transcription followed by polymerase chain reaction and the branched DNA assay, we detected HCV RNA in 75 patients coinfected with HIV and HCV and in 75 patients infected with HCV alone. The HIV RNA was also quantified by the branched DNA assay and the p24 antigenaemia was determined by enzyme-linked immunosorbent assay. The immune response to HCV was studied in the 150 patients by the use of third generation recombinant immunoblot assay (RIBA). RESULTS: Although a comparable number of patients had detectable HCV viraemia in both groups, HCV RNA was quantifiable in 79% of HIV-positive patients and in only 43% of HIV-negative patients (P < 10(-5)), and the mean HCV RNA level was much higher in the HIV-positive group than in the HIV-negative group (P < 10(-7)). The quantity of HCV RNA did not correlate with the CD4 count, p24 antigenaemia or HIV RNA level. The analysis of RIBA showed 14.7% indeterminate or negative results in the HIV-positive group and only 4% indeterminate results in the HIV-negative group. HIV-positive patients had reactivity to less antigen bands than HIV-negative patients (P < 10(-3)), and they had a weaker reactivity to c100, c33c and NS5 antigen bands than HIV-negative patients. CONCLUSION: Our results show that in the case of HIV infection, the HCV RNA levels are strongly increased, but HCV load is not linked to the immunosuppression induced by HIV; therefore, the present data do not support the hypothesis of a direct interaction between HIV and HCV.

Adolescent↗

PubMed2006

High-throughput screening of RNA polymerase inhibitors using a fluorescent UTP analog.

RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (gamma-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of gamma-AmNS-PPi, which has higher intrinsic fluorescence than (gamma-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 microM. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC(50) below 100 microM. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has >99% sequence identity with Mycobacterium tuberculosis RNAP subunits.

DNA-Directed RNA Polymerases↗

PubMed1991

Cytochrome c mRNA levels decrease in senescent rat heart.

The concentration of mitochondria decreases in the heart as rodents age from maturity to senescence. The reason for this change is not known. One purpose of the present study was to determine if cytochrome c mRNA, representative of proteins of the inner mitochondrial membrane, decreased in the hearts of Fischer 344 rats as they aged from 12 to 24 months. Twenty-two percent less cytochrome c mRNA existed per given quantity of extracted RNA from the heart in 24-month-old rats as compared with the 12-month-old group. No change in the quantities of cardiac alpha-actin mRNA, Ca2+/calmodulin protein kinase II mRNA or 18S rRNA was noted between 12- and 24-month-old hearts. Thus, the decrease in cytochrome c mRNA suggests that decreases in mRNAs for proteins of the inner mitochondrial membrane could play some role in the diminished concentration of mitochondria that exists in the senescent heart.

Actins↗

PubMed1983

Characterization and localization of calcitonin messenger ribonucleic acid in rat thyroid.

DNA/RNA hybridization assays have been used to examine calcitonin (CT) RNA production in normal rat thyroids. A cloned CT cDNA which codes for the entire rat CT precursor was radiolabeled to a high specific activity and used in hybridization assays to explore 1) the sizes and relative quantities of CT RNA extractable from thyroids obtained from rats of differing ages; 2) the effect of calcium on the in vitro production of CT RNA in rat thyroid tissue slices; and 3) the localization, by hybridization histochemistry, of C cells in rat thyroid that contain CT RNA. The relative concentrations of CT RNA, per microgram of total thyroid RNA, increased remarkably with age, with 14-month-old rats having approximately 14-fold elevated concentrations of thyroidal CT RNA compared to 19-day-old rat fetuses. Of interest was the finding that a second larger species of CT RNA is only evident in thyroids obtained from 14-month-old animals. The effect of calcium on the in vitro production of CT RNA in rat thyroid tissues was studied over 3- and 6-h periods. Although previous investigations have shown that calcium causes an immediate and linear increase in CT secretion from the thyroid gland, no net increase vs. controls in the amount of CT RNA extractable from calcium-stimulated thyroid slices was observed. Finally, hybridization histochemistry, a technique that identifies in fixed tissue sections those areas that contain a specific mRNA population, was used to localize C cells in the thyroid containing CT RNA. Specific areas of rat thyroid hybridized with the CT cDNA probe and autoradiography revealed these areas to be parafollicular cells located only in the central portion of the thyroid lobes, mRNA quantities detected by hybridization histochemistry showed little variation over the central area of the thyroid, indicating the C cells in this region of the thyroid are accumulating CT RNA at approximately the same rate.

Animals↗

PubMed1979

Expression of retrovirus-associated 8S RNA in mammalian cells.

A comparative study of the expression of retrovirus-associated 8S RNA was made in different mammalian cells. This RNA is found in cultured cells from all mammalian species analysed but its expression varies. An increase of 8S RNA is observed in sarcoma virus-transformed cells as compared to control uninfected cells or cells infected with leukaemia virus. No increased quantities of 8S RNA were detected in cells transformed by SV40 or by methylcholanthrene. These data show that the level of 8S RNA was augmented following transformation by sarcoma viruses.

Animals↗

PubMed1993

Effect of captopril on the cytological and biochemical changes induced by adriamycin.

Captopril, an angiotensin-converting enzyme inhibitor, was evaluated for its antimutagenic potential. Male Swiss albino mice (6-8 wk old) were treated orally with different doses of captopril dissolved in water for 7 days. Some of the mice in each group were injected ip with adriamycin (ADM; 15 mg/kg body weight) and killed after 30 hr. Femoral cells of mice were collected and studied for reduction of micronuclei. Proteins, RNA and DNA were determined in hepatic cells. Captopril pretreatment was found to reduce ADM-induced micronuclei in polychromatic cells and increase the quantity of protein, RNA and DNA in hepatic cells. The inhibition of clastogenicity observed may be due to free-radical scavenging action of captopril.

Administration, Oral↗

PubMed2005

Optimization of liver biopsy RNA sampling and use of reference RNA for cDNA microarray analysis.

In this study, we used the rat liver as a model system to optimize the conditions for extracting RNA from liver biopsies for use in cDNA microarrays. We found that a 5-mm biopsy with a 16-gauge needle and storage in RNA later at 4 degrees C were optimal conditions for RNA extraction. The most important factor for the quantity and quality of RNA extraction was the sample diameter. Using the optimized sampling conditions and a cDNA microarray, we compared the expression of genes in the normal and the fibrotic tissues of the LEC rat liver, a model of liver tumorigenesis, with SD rat liver RNA as a reference. We found 29 genes that were up-regulated and 33 genes that were down-regulated in the fibrotic part of the liver. Furthermore, with the help of the reference RNA, we were able to classify the expression profiles into five groups without complex mathematical analyses; without the reference RNA, the genes could be classified into only two groups. Finally, we found that osteopontin was expressed at a very high level in the fibrotic portion of the LEC rat liver. This cDNA microarray result was validated by immunohistochemistry, which showed an elevated expression of osteopontin in the region of cholangiocarcinoma and a lack of expression in normal tissues. With optimized conditions, we should be able to apply the microarray system for routine practice.

Animals↗

PubMed2006

Purification of plant viral and satellite double-stranded RNAs on DEAE monoliths.

Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue. The developed chromatographic method revealed the pathogens' presence in only 15 min, avoiding nucleic acid precipitation and electrophoretic analysis.

Chromatography, Liquid↗

PubMed1978

Terbium as a solid-state probe for RNA.

This paper continues previous work on the analysis of nucleic acid-terbium complexes in the solid state. The fluorescence excitation and emission spectra of the RNA-terbium(III) complex is reported. The fluorescence excitation and emission spectra of both the RNA-terbium(III) and DNA-terbium(III) complexes as trapped on millipore filters is reported. One hundred percent of the DNA combined with terbium was trapped on millipore filters. Deoxyribonucleic acid was recovered from DNA-terbium(III) complexes trapped on millipore filters using SDS-extraction. Energy transfer was shown to occur from the bases in nucleic acids to the terbium ion, whereas the actual binding of terbium to nucleic acids was due to phosphate groups. The relative fluorescence of homopolyribonucleotide-terbium complexes showed that the guanine moiety was responsible for most of the observed fluorescence. Binding studies showed an equal affinity of radioactive terbium for all the homopolyribonucleotides. The fluorescence of solid-state DNA and RNA terbium complexes was used to measure picomole quantities of DNA or RNA.

Animals↗

PubMed2004

Effect of formalin on extraction of mRNA from a formalin-fixed sample: a basic investigation.

BACKGROUND: The validity of molecular studies using RNA extracted from decades-old formalin-fixed and embedded tissue blocks is well documented. Formalin-fixed samples are, however, known to be poor materials for molecular biological applications. The aim of this study was to investigate the effect of formalin as a preservation agent on RNA quality and relevant molecular applications. METHODS: The quantity and quality of RNA from liver tissues are modified by the fixation time. Rat liver tissues were harvested and fixed for 4, 8, 12, and 24 h in 10% buffer formalin, and only 9% of RNA could be extracted from the formalin-fixed tissue stored for 4 h. RESULTS: Agarose gel electrophoresis did not show the characteristic pattern of RNA with 18S and 28S in all lanes. Based on the results, it is thought that the RNA was damaged owing to one or more of the following reasons: 1) formalin directly degrades RNA, 2) formalin inactivates RNase, and/or 3) formalin destroys the reagent for extraction of RNA. In this study, it was shown by electrophoresis that very little RNA was extracted by Trizol with 1% formalin. CONCLUSIONS: Formalin has direct and indirect ill effects on RNA during the extraction process.

Animals↗