Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA quantity”

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 235 records · Page 13Linked to original sources
PubMed2000

Use of SMART-generated cDNA for differential gene expression studies.

A major limitation of gene expression profiling using microarrays has been the substantial amount of RNA required for standard probe labeling techniques, especially when working with clinical samples such as biopsies, microdissected tumors, and laser-captured cells. CLONTECH's PCR-based SMART technology (Switch Mechanism At the 5'end of RNA Templates) bypasses this problem by allowing accurate cDNA amplification from nanogram quantities of total RNA. This amount of RNA ensures that SMART amplification yields a pool of cDNA, which reflects the sample's original complexity and relative abundance of the original RNA sample.

Cell Separation↗
PubMed1993

[Schistosoma japonicum adult worm RNA isolation with Chomczynski's method].

A rapid method of total RNA of Schistosoma japonicum adult worm isolation by single step extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of intact RNA and can be completed within several hours. It is particularly useful for isolation of RNA from minute quantity of parasite material. Analysis of agarose gels electrophoresis reveals that the RNA preparations show a distinct band comigrating with 18S rat brain RNA and without large rRNA species. The large rRNA is in vivo nicked. The absence of a band at approximately 26S presumably reflects post transcriptional processing of the large rRNA.

Animals↗
PubMed1978

Direct demonstration of immunoglobulin kappa chain RNA in thymus T cells by in situ hybridization.

Mouse thymuses with more than 99% T cells have been reported to contain immunoglobulin kappa mRNA-like molecules (kappa RNA) in relatively large quantities. The present study was undertaken to rule out the possibility that the kappa RNA was mainly a product of a few contaminating B cells of the thymus and to determine whether all T-cell subpopulations contained kappa RNA. By in situ hybridization with DNA complementary to kappa mRNA (kappa cDNA) the following observations were made: 98.5% of thymus cell preparations hybridized with kappa cDNA; the 1.5% unlabeled cells were generally larger and paler staining than the majority of thymus cells. Only 0.015% of thymus cells were intensely labeled and appeared to be plasma cells. Also, 87% of spleen cells hybridized with kappa cDNA; most of these showed similar labeling intensity to the majority of thymus cells. The number of unlabeled cells corresponded to the percentage of hemopoietic cells and macrophages in the spleen. Spleen cells in the range of 0.37-0.85% were intensely labeled and appeared to be plasma cells. The following controls supported the conclusion that the results with thymus and spleen were due to specific hybridization: most of the kappa mRNA-deficient tissue culture cells of the plasmocytoid tumor ABPL-4 did not hybridize with kappa cDNA. The kappa mRNA-producing cells from myeloma PC 3741 hybridized in situ with kappa cDNA. Furthermore, all cells from this tumor and all spleen cells hybridized uniformly with a cDNA probe complementary to most of the total cellular poly(A)-containing RNA species of these cells. These results indicate that T cells of all types in the thymus as well as in the periphery contain substantial quantities of kappa RNA.

Animals↗
PubMed1990

Silkmoth chorion antisense RNA. Structural characterization, developmental regulation and evolutionary conservation.

Choriogenic follicular cells of the silkmoth Bombyx mori contain significant quantities of antisense RNA transcribed from chorion genes. Antisense RNA derived from a chorion gene with a high content of cysteine, HcB.12, was characterized in detail. The antisense transcripts are initiated downstream from the 3' end of HcB.12 mRNA and extend over 75% of the length of the gene, comprising its entire second exon and part of its intervening sequence. The antisense RNA is devoid of any significant open reading frames and is not polyadenylated. These features, combined with the presence of specific sequence motifs within its transcribed and upstream region, suggest that antisense RNA may be transcribed by RNA polymerase III. Chorion antisense RNA is detectable only in choriogenic follicular cells and appears to be co-ordinately regulated with chorion mRNA. Its cytoplasmic accumulation during choriogenesis parallels that of the corresponding mRNA. Although chorion mRNA is at least five times more abundant than antisense RNA, the latter is present as a single-stranded entity in follicular cytoplasm but can form perfect duplexes with its mRNA complement upon annealing in vitro. The possible involvement of antisense RNA transcription in the pathway that controls the programmed expression of chorion genes at the level of transcription initiation or post-transcriptional processing is discussed.

Animals↗
PubMed1989

Characterization of single base substitutions in edited apolipoprotein B transcripts.

Mature RNA transcripts from a single eukaryotic gene may contain different nucleotide sequences, ranging from alternately spliced exons to transcripts from separate alleles differing by only one base. Our laboratory and others have recently reported another class of RNA sequence differences, occurring in transcripts from the single copy apolipoprotein B (apoB) gene. A unique RNA editing mechanism allows expression of the CAA glutamine codon encoded by the apoB gene at nucleotide 6666, or terminates translation by the introduction of a premature UAA translational stop codon. In this study, we used the polymerase chain reaction (PCR) to amplify and characterize edited apoB RNA transcripts differing by a single nucleotide. Amplification and sequence analysis from small quantities of total RNA will facilitate the study of RNA editing and transcription in general.

Animals↗
PubMed1972

Transcription during the development of bacteriophage phi 29: production of host- and phi 29-specific ribonucleic acid.

The synthesis of ribonucleic acid (RNA) during development of the virulent Bacillus subtilis bacteriophage phi29 has been analyzed. Transcription of host deoxyribonucleic acid (DNA) continues at the preinfection rate throughout the latent period of viral growth. RNA-DNA hybridization was used to show that host messenger RNA synthesis continues late into the phage lytic cycle. Amino acid-labeling experiments show that this RNA is continuously used to produce protein. Ribosomal RNA production is not inhibited by phage infection. Small quantities of phage-specific RNA first appear between min 6 and 9 after infection. This RNA is made exclusively from one of the phi29 DNA strands. At 12 min postinfection, when phage DNA replication commences, large quantities of viral RNA start to be synthesized. This RNA appears to be transcribed from both strands of phi29 DNA. Studies with rifamycin and rifamycin-resistant host strains showed that the production of all phage phi29-specific RNA requires those components of the host RNA polymerase which are sensitive to this antibiotic. Thus, phage phi29 does not stop transcription of host DNA and may produce only one element for regulation of transcription of its own DNA. These findings may reflect the limited amount of genetic information carried by this phage.

Autoradiography↗
PubMed2002

The HCV and HIV coinfected patient: what have we learned about pathophysiology?

Hepatitis C virus (HCV) infection is an important problem in individuals who are also infected with HIV. HCV infection is very common in HIV-infected individuals, occurring in approximately one quarter to one third of this group, presumably as a consequence of shared routes of transmission related to virologic and pathogenic aspects of the viral infections. Although both are single-stranded RNA viruses and share similar epidemiologic properties, there are many important differences. Although the quantity of HIV RNA in plasma is an important prognostic determinant of HIV infection, this has not been shown with HCV. A direct relationship is apparent between HIV-related destruction of CD4 cells and the clinical consequences of the disease resulting from immunodeficiency. The pathogenesis of HCV, which occurs as a consequence of hepatic fibrosis, is much more complex. The hepatic stellate cell, the major producer of the extracellular matrix protein, is the main contributor to hepatic fibrosis, but the mechanism by which HCV induces hepatic fibrosis remains unclear. Treatment of HCV is increasingly important in HIV-infected patients due to improved HIV-associated morbidity and mortality and due to the frequency with which HCV occurs in patients with HIV-HCV coinfection. Timing of treatment initiation, management of side effects, and possible effects of anti-HCV therapy on HIV are among the issues that need consideration. Also, because several issues concerning HCV are unique to coinfected patients, further research is needed to determine optimal management of HCV in this setting.

HIV Infections↗
PubMed2003

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts.

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.

Gene Expression Profiling↗
PubMed1994

The impact of hepatitis C virus infection on renal allograft recipients.

A second generation hepatitis C virus recombinant immunoblot assay (RIBA) was used to screen stored perioperative serum from 641 renal allograft recipients. One hundred and nine (17%) were anti-HCV positive at the time of transplant. RIBA positivity was found to be an independent predictor of post-transplant liver disease in a logistic regression model (P < 0.05). Moreover, RIBA positive patients were at greater risk for infectious events (P = 0.03) and rejection episodes (P = 0.002). The cumulative dose of antilymphoblast globulin administered as induction therapy was an independent predictor of post-transplant liver disease in a dose response relationship. Qualitative PCR showed that 74% of the perioperative RIBA positive patients had detectable HCV RNA in a current serum sample. Further, quantitative HCV RNA analysis with a competitive template PCR and HCV strain identification by restriction fragment length polymorphism demonstrated a large range of HCV RNA copies/ml of serum and three different HCV strains (BK, Hutch and HCV-1). Neither quantity of HCV RNA nor strain type correlated with abnormal transaminases post-transplant. As yet, there has not been an effect of anti-HCV status on actuarial patient and graft survival. This study suggests that anti-HCV is not a contraindication to renal transplantation; however, we would recommend that the pre-transplant evaluation of the anti-HCV positive patient include a liver biopsy to properly stage the disease. Close post-transplant follow-up is required in view of the increased risk for infection and rejection.

Adult↗
PubMed1979

Mouse mammary tumor virus genome expression in chemical carcinogen-induced mammary tumors in low- and high-tumor-incidence mouse strains.

Involvement of mouse mammary tumor virus (MMTV) in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis was investigated in low- (BALB/c) and high- (BALB/cfC3H) mammary-tumor-incidence mouse strains. Both strains contain endogenous MMTV integrated into the cellular genome. Additionally, BALB/cfC3H mice are infected with exogenous MMTV-S which is responsible for a higher incidence of mammary tumors in breeding females. Administration of DMBA to virgin mice of both strains resulted in a moderate frequency of mammary tumors within 40 wk after treatment. No differences were found in DMBA-induced tumor incidences at 18 wk (6% and 7%) or at 38 wk (29% and 36%) after treatment of BALB/c and BALB/cfC3H mice, respectively. Expression of MMTV in these tumors was examined by assaying for the presence of MMTV RNA by hybridization using MMTV-specific cDNA and by immunohistochemical staining utilizing antibodies against MMTV 52,000-dalton glycoprotein, gp52, and 28,000-dalton internal protein, p28. Of 16 BALB/c tumors assayed, 11 did not contain detectable levels of MMTV RNA and the remaining 5 tumors contained only low levels (0.0005-0.0010%) of viral RNA. Importantly, MMTV RNA was not detected in 5 of 27 BALB/cfC3H tumors. The other BALB/cfC3H tumors contained quantities of MMTV RNA ranging from 0.0006 to 0.4170%. Most BALB/cfC3H tumors with detectable levels of MMTV RNA also synthesized viral proteins gp52 and p28. Thus, expression of the complete MMTV genome is not requisite for maintenance of the tumor phenotype in DMBA-induced mammary tumors in either BALB/c or BALB/cfC3H virgin mice under 1 year of age.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

[Quantitative detection of SARS-CoV RNA in excreta and oropharyngeal washing fluid from convalescence patients with severe acute respiratory syndrome].

OBJECTIVE: To detect the severe acute respiratory syndrome coronavirus (SARS-CoV) RNA in excreta and oropharyngeal washing fluid (OWF) from the convalescence SARS patients, and to determine whether convalescence patients carry the SARS-CoV, and whether having infectivity. METHODS: Totally 531 samples (including urine, stool, and OWF) were collected from 177 convalescence patients with positive SARS antibody which were confirmed by ELISA method. Real-time quantitative PCR was performed to detect the RNA of the SARS-CoV, and results were analyzed by SPSS analysis software. RESULTS: There were 49 (27.7%) cases of SARS-CoV RNA detection positive in 177 patients, including 31 (17.5%) cases with one sample positive, 14 (7.9%) cases with two samples positive, and 4 (2.3%) cases with three samples positive. The positive rates of urine, stool, and OWF were 14.7% (26/177), 11.9% (21/177), and 13.6% (24/177), respectively. The quantity of SARS-CoV RNA in samples was 100-47 000 copies/ml. No significant difference was found among urine, stool, and OWF on the difference grade quantity of SARS-CoV RNA quantity. CONCLUSIONS: About 10% convalescence SARS patients might still carry the SARS-CoV in hospitalization. The detection of SARS-CoV RNA by real-time quantitative PCR may become a laboratory examination warranty for convalescence SARS patients to discharge hospital and relieve separation.

Adolescent↗
PubMed1998

Cloning differentially regulated genes from chondrocytes using agarose gel differential display.

The technique of RNA differential display has been used extensively to clone differentially expressed genes from a wide variety of cells and tissues. Recently, a simplified method of cloning differential display products, separated on agarose gels, was described. Here we report an adaption of this method, using total RNA, to clone differentially expressed genes. The approach is simple and rapid, and requires only small quantities of total RNA. Utilising this approach, we have cloned three differentially regulated genes from chondrocytes stimulated to hypertrophy in vitro, and confirmed their pattern of expression by Northern blotting. These gene fragments were sequenced and found to correspond to known genes, although only one has previously been isolated from chondrocytes.

Animals↗
PubMed1989

Long-term effect of elevated temperatures on SSPE virus expression in persistently infected rat glial cells.

Cultivation of measles virus (SSPE virus, Lec strain) persistently infected C6 rat glioma cells at 39 degrees C resulted in the loss of detectable expression of measles virus proteins. Temperature shift-back led to reactivation of measles virus even after maintenance of the cells at 39 degrees C for 15 days. In Northern blot analysis viral mRNA disappeared at 3 days after shift-up whereas 50 S viral genome-sized RNA was detectable until 6 days. The 50 S RNA decreased in quantity in rough correlation with dilution by cell passage at 39 degrees C. The 50 S viral RNA was found in the nucleocapsid fraction. On day 9 after shift-down of persistently infected cells, maintained at 39 degrees C for 15 days, 50 S viral RNA reappeared although mRNAs were not yet detected. Infectious center assays showed that the number of cells in the population at 39 degrees C, which contained an SSPE virus genome that could be reactivated, declined after temperature shift. Moreover, cell cloning experiments, in which single cells of cultures maintained for various lengths of time at 39 degrees C were incubated at 35 degrees C and examined by immunofluorescence, reconfirmed the above results. This indicates that the reactivation of SSPE virus described here was due to re-infection of virus-antigen negative cells with progeny virus produced by a few latently infected cells in the population. The biological significance of this phenomenon in the central nervous system virus infection is discussed.

Animals↗
PubMed1994

Measurement of mRNA for E-selectin, VCAM-1 and ICAM-1 by reverse transcription and the polymerase chain reaction.

Stimulation of cultured human umbilical vein endothelial cells by cytokines such as interleukin-1 and tumour necrosis factor induces de novo synthesis and expression of the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In general, alterations in cell surface expression of these molecules are known to be related to increased gene transcription and altered levels of mRNA. The extension of these observations to the study of inflammatory processes in different human organs necessitates the development of techniques for the quantification of mRNA in small tissue samples. Here we present a method for the quantification of mRNA for E-selectin, VCAM-1 and ICAM-1 using reverse transcription and the polymerase chain reaction (RT-PCR). For each molecule of interest a mutant RNA was synthesised consisting of the wild-type sequence deleted of 15-20 bases. The mutant and wild-type RNA sequences are recognised by the same primers, and can therefore be amplified competitively in the same tube by RT-PCR. As the mutant and wild-type RNAs compete for the primers, the amount of wild-type RNA can be determined by the size of the dominant product that results after addition of known quantities of mutant RNA. Using this detection and quantification method we have examined the dose dependency and time course of mRNA accumulation following TNF-alpha stimulation of HUVEC. Similar time-courses of E-selectin, ICAM-1 and VCAM-1 mRNA accumulation were observed by competitive RT-PCR as by laser densitometry of Northern blots. Finally we were able to show that the technique could measure changes in levels of mRNA for these three molecules in human skin biopsies taken at different times during the development of a delayed hypersensitivity response to tuberculin purified protein derivative. This technique should be useful for the study of adhesion molecule mRNA in small tissue culture samples and in biopsies.

Base Sequence↗
PubMed1979

Relationship between RNA content and progression of lymphocytes through S phase of cell cycle.

A flow cytometric technique for simultaneous measurements of RNA and DNA in individual cells has been applied to correlate the content of cellular RNA with the rate of progression of cells through the S phase. Human peripheral lymphocytes stimulated with phytohemagglutinin were blocked at the G1/S phase boundary by hydroxyurea or 5-fluorodeoxy-uridine treatment. Cells in the G1 phase as well as cells blocked at the G1/S phase boundary showed high heterogeneity with respect to stainable RNA content. After release from the block, the cells traversed the S phase at rates proportional to the quantity of stainable RNA per cell. Cells with the highest RNA content completed DNA replication 5 hr after release from the block; the cells with minimal RNA traversed the S phase at one-fifth of this rate. The large intercellular variation in stainable RNA and length of the S phase may be due to functional heterogeneity in the lymphocyte population. Our results suggest a correlation between the number of ribosomes and the rate of DNA replication in lymphocytes.

Cell Cycle↗
PubMed2006

Expression of 4 genes between chromosome 15 breakpoints 1 and 2 and behavioral outcomes in Prader-Willi syndrome.

Prader-Willi syndrome is a neurodevelopmental disorder that is characterized by infantile hypotonia, feeding difficulties, hypogonadism, mental deficiency, hyperphagia (leading to obesity in early childhood), learning problems, and behavioral difficulties. A paternal 15q11-q13 deletion is found in approximately 70% of patients with Prader-Willi syndrome, approximately 25% have uniparental maternal disomy 15, and the remaining 2% to 5% have imprinting defects. The proximal deletion breakpoint in the 15q11-q13 region occurs at 1 of 2 sites located within either of 2 large duplicons allowing for the identification of 2 deletion subgroups. The larger, type I (TI) deletion involves breakpoint 1, which is close to the centromere, whereas the smaller, type II (TII) deletion involves breakpoint 2, located approximately 500 kilobases distal to breakpoint 1. Breakpoint 3 is located at the distal end of the 15q11-q13 region and common to both typical deletion subgroups. Analyses of the genetic subtypes of Prader-Willi syndrome to date have primarily compared individuals with typical deletion and uniparental maternal disomy 15 without grouping the individuals with a deletion into TI or TII. Distinct differences have been reported between individuals with Prader-Willi syndrome resulting from deletion compared with uniparental maternal disomy 15 in physical, cognitive, and behavioral parameters. We previously presented the first assessment of clinical differences in individuals with Prader-Willi syndrome categorized as having type I or II deletions. Adaptive behavior, obsessive-compulsive behaviors, reading, math, and visual-motor integration assessments were generally poorer in individuals with Prader-Willi syndrome and the TI deletion compared with subjects with Prader-Willi syndrome with the TII deletion or uniparental maternal disomy 15. Four genes (NIPA1, NIPA2, CYFIP1, and GCP5) have been identified in the chromosomal region between breakpoints 1 and 2 and are implicated in compulsive behavior and lower intellectual ability observed in individuals with Prader-Willi syndrome with TI versus TII deletions. We quantified messenger-RNA levels of these 4 genes in actively growing lymphoblastoid cells derived from 8 subjects with Prader-Willi syndrome with the TI deletion (4 males, 4 females; mean: age 25.2 +/- 8.9 years) and 9 with the TII deletion (3 males, 6 females; mean age: 19.5 +/- 5.8 years). Messenger-RNA levels were correlated with validated psychological and behavioral scales administered by trained psychologists blinded to genotype status. Messenger RNA from NIPA1, NIPA2, CYFIP1, and GCP5 was reduced but detectable in the subjects with Prader-Willi syndrome with the TI deletion, supporting biallelic expression. For the most part, messenger-RNA values were positively correlated with assessment parameters, indicating a direct relationship between messenger-RNA levels and better assessment scores, with the highest correlation for NIPA2. The coefficient of determination indicated the quantity of messenger RNA of the 4 genes explained from 24% to 99% of the variation of the behavioral and academic parameters measured. By comparison, the coefficient of determination for deletion type alone explained 5% to 50% of the variation in the assessed parameters. Understanding the influence of gene expression on behavioral and cognitive characteristics in humans is in the early stage of research development. Additional research is needed to identify the function of these genes and their interaction with gene networks to clarify the potential role they play in central nervous system development and function.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Purification of histidine-tagged T4 RNA ligase from E. coli.

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.

Bacteriophage T4↗
PubMed2006

Detection of biomarker gene expression by real-time polymerase chain reaction using amplified ribonucleic acids from formalin-fixed random periareolar fine needle aspirates of human breast tissue.

OBJECTIVE: To address the detection of breast cancer biomarker gene expression in formalin-fixed random periareolar fine needle aspiration (RPFNA) samples of benign breast tissue collected during breast cancer prevention trials by quantitative real-time polymerase chain reaction (qPCR). STUDY DESIGN: Formalin-fixed breast epithelial cells collected by RPFNA and processed as thin layer preparations were isolated by laser capture microdissection (LCM). Ribonucleic acid (RNA) was extracted and amplified using a single round of T7-based linear amplification followed by quality assessment and biomarker assay using TaqMan chemistry. RESULTS: More than 80% of RPFNA samples yielded RNA of sufficient quantity and quality for measurement of a panel of biomarker genes following a single round of linear amplification. RNA and protein expression for estrogen receptor alpha, as assessed by LCM/qPCR and immunohistochemistry, were correlated. Amplification plots were similar for cDNA standards and cDNA derived from RPFNA samples. CONCLUSION: Assessment of gene expression using amplified RNA from microdissected formalin-fixed RPFNAs can increase the number of biomarkers used during breast cancer chemoprevention trials.

Biomarkers↗