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At least 1,099 records · Page 61Linked to original sources
PubMed2003

Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate.

Rapid extraction of total RNA from Eucalyptus leaves is difficult due to the high content of polyphenolics and polysaccharides. A rapid and simple method was developed by using an extraction buffer containing sodium isoascorbate at a concentration of 500 mM. This method consisted of one or two chloroform extractions, one acid guanidium-phenol-chloroform extraction, and isopropanol precipitation alone. The yields of the RNA fractions were 246-1750 micrograms/g fresh weight when leaves of Eucalyptus, five other woody plants, and four herbaceous plants were used as samples. The contamination of the RNA fractions by proteins and polysaccharides was very limited as judged spectrophotometrically. When the RNA fractions were subjected to agarose gel electrophoresis, intact rRNA bands were detected. The RNA fractions could be used for RT-PCR. These results indicate that our new method achieves a simple and rapid preparation of high-quality RNA from leaves of Eucalyptus and other plant species.

Ascorbic Acid↗
PubMed1996

Failure to detect paramyxovirus RNA in the skin of connective tissue disease.

Etiology of connective tissue disease is unknown. The association of infectious agents has been suspected serologically, ultrastructurally and recently by means of molecular biological techniques. We extracted RNA from lesions of 25 discoid lupus erythematosus, 9 systemic LE, and 3 systemic sclerosis biopsies, and reverse transcription-polymerase chain reaction was performed for all paramyxoviruses known to infect human beings. None of the samples yielded positive signal for any of the viruses. The existence of paramyxovirus in the skin of connective tissue disease is very unlikely.

Base Sequence↗
PubMed2005

Oral treatment with PD-0200347, an alpha2delta ligand, reduces the development of experimental osteoarthritis by inhibiting metalloproteinases and inducible nitric oxide synthase gene expression and synthesis in cartilage chondrocytes.

OBJECTIVE: To examine the in vivo effects of PD-0200347, an alpha(2)delta ligand of voltage-activated Ca(2+) channels and a compound chemically related to pregabalin and gabapentin, on the development of cartilage structural changes in an experimental dog model of osteoarthritis (OA). The effects of PD-0200347 on the major pathways involved in OA cartilage degradation, including matrix metalloproteinases (MMPs) and the inducible form of nitric oxide synthase (iNOS), were also studied. METHODS: OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were randomly distributed into 3 groups and treated orally with either 1) placebo, 2) 15 mg/kg/day of PD-0200347, or 3) 90 mg/kg/day of PD-0200347. Dogs were killed 12 weeks after surgery. The severity of the lesions was scored macroscopically and histologically. Cartilage specimens from the femoral condyles and tibial plateaus were processed for RNA extraction and quantitative reverse transcription-polymerase chain reaction (RT-PCR) or immunohistochemistry. Specific probes and antibodies were used to study the messenger RNA and protein levels of iNOS, MMP-1, MMP-3, and MMP-13. RESULTS: No clinical signs of drug toxicity were noted in the treated animals. Treatment with PD-0200347 at both dosages tested (15 and 90 mg/kg/day) reduced the development of cartilage lesions. There was a reduction in the score of lesions, with a statistically significant (P = 0.01) difference when the highest dosage of the drug was administered. The reduction in the score was mainly related to a decrease in the surface size of the lesions. Quantitative RT-PCR showed that PD-0200347 significantly reduced the expression of MMP-13, a key mediator in OA. Immunohistochemical analyses showed that treatment with PD-0200347 significantly reduced the synthesis of all key OA mediators studied. CONCLUSION: This study demonstrated the efficacy of PD-0200347 in reducing the progression of cartilage structural changes in a dog model of OA. It also showed that this effect is linked to the inhibition of the major pathophysiologic mediators responsible for cartilage degradation.

Administration, Oral↗
PubMed1995

Quantitative analysis of HIV-1 RNA in plasma preparations.

HIV-1 RNA extraction methodology, stability and cellular location in plasma were studied by quantitative analysis using reverse transcriptase (RT) and polymerase chain reaction (PCR). HIV-1 RNA as intact virus was stable in plasma at room temperature for at least for 24 h, or stable in RNAzol (Tel-Test, Inc. Texas) at -70 degrees C for at least 6 months. The HIV-1 RNA PCR signal did not decline significantly after freezing and thawing of the virus in plasma or in RNAzol. To assess the effect of plasma constituents from different individuals upon quantitative PCR, identical copy members of HIV LAI were spiked into plasma from 9 different, normal individuals. PCR detection of HIV-1 RNA did not show any significant variation in quantitative signals. Additionally, platelet-rich plasma from three seropositive subjects was fractionated into a platelet-free plasma fraction and a platelet pellet fraction. The quantitative analysis of HIV-1 RNA in these fractions, and in the corresponding peripheral blood lymphocytes (PBLs) from each patient, demonstrated that the majority of the HIV-1 RNA was distributed in the plasma, and the HIV-1 RNA in the plasma of these patients seemed not to be strongly platelet associated.

Blood Platelets↗
PubMed1994

The detection of tomato spotted wilt virus using the polymerase chain reaction.

A polymerase chain reaction (PCR) based assay was used to detected a number of UK tomato spotted wilt virus (TSWV) isolates in total RNA extractions made from infected plant material. Extracts were reverse transcribed and the resultant cDNA amplified by PCR, using oligonucleotide primers specific for a 276 base pair fragment of the L RNA segment. Assay products were electrophoresed on agarose gels and visualised by ethidium bromide staining. The viral origin of the product produced was confirmed by sequencing, with the data obtained having very high homology with previously published L RNA sequence data. The specificity and sensitivity of the RT-PCR assay, in comparison with existing tests, is discussed.

Base Sequence↗
PubMed2005

Standardization strategy for quantitative PCR in human seminoma and normal testis.

Housekeeping genes are commonly used as endogenous references in quantitative RT-PCR. Ideally these genes are constitutionally expressed by all cell types and do not vary under experimental conditions. Tissues of 9 normal testes and 22 classical pure seminoma were obtained for RNA-extraction. Real-time RT-PCR was used to examine the mRNA-expression of ubiquitin C, beta-actin, GAPDH, 18S ribosomal RNA (18S rRNA) and porphobilinogen-deaminase (PBGD). Additionally, 3 normal testicular tissues and 39 seminoma, including 1 normal testis and 17 seminoma of the RT-PCR group, were utilized for microarray analyses. Ubiquitin C (protein degradation) was down-regulated, GAPDH (carbohydrate metabolism), beta-actin (cytoskeleton), 18S rRNA (ribosome) and PBGD (porphyrin metabolism) were up-regulated in seminoma. A normalization of the target gene data with up-regulated housekeeping genes would equalize or underestimate up-regulated data and overestimate down-regulated data. We demonstrate that none of the investigated housekeeping genes is suitable for normalization of the target gene RT-PCR data, but may be essential for tumor metabolism in human seminoma. Further, we developed a standardization strategy, which is applicable to many experimental investigations.

Biomarkers, Tumor↗
PubMed1998

Construction of internal cDNA competitors for measuring IL-10 and IL-12 cytokine gene expression in swine.

A competitive PCR assay (cPCR) was used to quantify swine cytokine responses to parasite infection. Internal standards (deleted cDNA competitor molecules [DcDNA mimics]) were produced and tested for swine interleukin-12 (IL-12), interleukin-10 (IL-10) and hypoxanthine phosphoribosyltransferase (HPRT) from PCR generated cDNA cloned in plasmid vectors. Deletion clones for the cDNA competitor molecules (DcDNA mimics) were generated for IL-10, IL-12 and HPRT by PCR in a single step and verified by (1) amplification of the expected smaller PCR product with the original primers, (2) appropriate fragment size released by restriction digestion of the deleted clone, and (3) correct sequence of the new DcDNA insert. DcDNA mimics were used to quantitate cytokine gene mRNA production during experimental and natural infections of swine with the gastrointestinal nematode parasite Trichuris suis. Mesenteric lymph node cells were collected from control and infected pigs at the time of maximal pathogenicity (35 days after infection) and snap frozen. After RNA extraction, samples were reverse transcribed (RT) to cDNA. cPCR was performed using the housekeeping gene HPRT DcDNA mimic and HPRT specific primers to insure RNA integrity and concentration. Cytokine cDNA content in these samples was then quantitated using cytokine mimics and gene specific primers. IL-10 gene expression in MLN draining the colon of pigs experimentally infected with T. suis increased 10-20 fold at day 35 compared to control pigs. IL-12 gene expression was not detectable in MLN of these pigs, but was detectable in MLN of pigs exposed naturally to T. suis on a contaminated dirt lot that also exhibited signs of secondary bacterial invasion. Swine IL-10 and IL-12 gene expression can be quantitated in local mesenteric tissues. This cPCR assay will enable scientists to quantitate cytokine gene expression in swine and determine the nature of immune responses to important infectious diseases.

Animals↗
PubMed1984

Elastin mRNA levels during foetal development of sheep nuchal ligament and lung. Hybridization to complementary and cloned DNA.

Elastin mRNA levels were quantified in sheep nuchal ligament and lung during the latter half of foetal development with elastin-specific cDNA (complementary DNA) probes using both hybridization in solution (saturation analysis) and hybridization on a fixed support (Northern analysis). For the solution-hybridization studies, cDNA prepared from nuchal-ligament mRNA was enriched to 65% for elastin sequences by hybridizing it to its template at a R0t (mol X s X litre-1) value that included only the abundant class of mRNA sequences. Hybridization of this probe to RNA extracted from nuchal ligament between 70 and 138 days after conception demonstrated elastin sequences increased about 10-fold (from 0.047 to 0.438% of total RNA). In contrast, lung elastin mRNA levels increased only 3-fold (from 0.009 to 0.022% of total RNA) during the same period. Over this development period these values correspond to increases in the average number of elastin mRNA molecules from 950 to 20 000 molecules/ligament cell and from 130 to 330 molecules/lung cell. For Northern analysis, elastin mRNA was purified from near-term-sheep nuchal ligament on sucrose density gradients. Analysis of the translation products of this elastin mRNA showed that relative elastin precursor synthesis was at least 80% of total [3H]valine incorporation. The Mr of this elastin mRNA, determined by methylmercury-agarose-gel electrophoresis, was approx. 1.25 X 10(6). Northern hybridization of nuchal ligament and lung RNA to a [32P]cDNA probe, transcribed from this sucrose-gradient-purified elastin mRNA, confirmed the developmental changes in elastin mRNA levels detected by solution-hybridization techniques. The specificity of this method was confirmed by using a cloned elastin gene fragment. These studies demonstrate that elastin mRNA levels in organs such as nuchal ligament and lung increase with foetal development, but that there are significant differences in the average cellular elastin mRNA content of these two organs.

Animals↗
PubMed1977

Analysis of polyoma virus nuclear RNA by mini-blot hybridization.

The size and sequence composition of virus-specific RNA extracted from the nuclei of mouse cells late during polyoma virus productive infection were studied by blot-hybridization analysis of 32P-labeled RNA fractionated on CH3HgOH/agarose gels. Viral RNA molecules between approximately 0.4 and 4 times the length of a complete transcript of the 5.4-kilobase circular viral DNA were found. Less than 20% of such molecules were polyadenylylated. Although viral RNA of all sizes contained species that together hybridized to the entire polyoma genome, sequences complementary to the late region were more abundant than sequences complementary to the early region in transcripts less than 10-12 kilobases long.

Base Sequence↗
PubMed2005

Optimizing microarray in experimental hypertension.

BACKGROUND: Genetic noise between outbred animals can potentially be a major confounder in the use of microarray technology for gene expression profiling. The study of paired organs from the same animal offers an alternative approach (e.g., for studies of the kidney in experimental hypertension). The present study was undertaken to determine the level of genetic noise between outbred adult Sprague-Dawley (SD) rats, and to determine the effects of unilateral nephrectomy on changes in gene expression as a basis for the design of microarray studies in experimental hypertension. METHODS: Male SD rats (approximately 130 g) were acclimatized before measurement of tail-cuff systolic blood pressure (SBP) for 6 control days and 4 days of saline treatment. Left kidney nephrectomy was performed, and the tissue snap-frozen in liquid nitrogen for subsequent RNA extraction. Two weeks later, SBP was measured over 4 control and 8 saline treatment days, and the remaining right kidney removed and frozen. Total RNA purification, preparation of cRNA, hybridization, and scanning of the Rat U34A Affymetrix arrays were performed, and data analyzed using MAS5 software Affymetrix Suite (v5), Bioconductor, as well as statistical methods motivated by relevant simulations. RESULTS: Gene expression profiles in the left control kidney were extremely consistent across animals. The expression profiles of pairs of kidneys from the same animal were, however, more similar than those of kidneys from different animals. Nephrectomy had little effect on the gene expression profiles in the time frame examined. CONCLUSION: Despite the outbred nature of the rats used in this study, they are useful for gene expression profiling comparisons. The use of paired organs from an individual animal ensures even further genetic identity, allowing determination of genes modified by the treatment of interest.

Animals↗
PubMed1974

Purification and biophysical properties of acute haemorrhagic conjunctivitis virus.

The buoyant density of acute haemorrhagic conjunctivitis virions labeled with either [(3)H]uridine or [(3)H]leucine was 1.34 g/ml in CsCl and 1.25 g/ml in sucrose. RNA extracted from the virions gave a sedimentation coefficient of approximately 34S in sucrose, and was found to be sensitive to RNase. Molecular weight of RNA was calculated to be 2.5 x 10(6) using poliovirus RNA for reference.

Centrifugation, Density Gradient↗
PubMed1968

Cellular origin of a mouse leukemia viral ribonucleic acid.

Mouse erythroblastosis virus, a member of the mouse leukemia virus group, was obtained from chronically infected C(3)H mouse embryo cells and purified on sucrose gradients. The ribonucleic acid (RNA) extracted from ribonuclease-treated virus consisted of a rapidly sedimenting (72S) species and a more slowly sedimenting component (4 to 30S). The 72S RNA did not contain base sequences homologous to deoxyribonucleic acid (DNA) from infected cells as determined by hybridization studies. In contrast, the slowly sedimenting RNA enclosed within the virus had base sequences homologous to DNA from infected and uninfected C(3)H mouse embryo cells.

Animals↗
PubMed1979

Viral genome RNA serves as messenger early in the infectious cycle of murine leukemia virus.

When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts.

Animals↗
PubMed1999

Evidence that the genomic RNA of hepatitis E virus is capped.

Hepatitis E virus (HEV) is an unclassified virus with a positive-sense RNA genome and an undefined replication strategy. In order to determine whether the HEV genome is capped or not, we developed a reverse transcription-PCR assay that is based on the ability of a monoclonal antibody to recognize 7-methylguanosine (m(7)G). Antibody to m(7)G bound RNA extracted from virions of two different HEV genotypes. The cap analog competitively inhibited the binding of virion RNAs, demonstrating that HEV has a capped RNA genome.

Genome, Viral↗
PubMed2006

Expression of interleukin 11 and its receptor and their prognostic value in human breast cancer.

BACKGROUND: Recent experimental evidence has shown a potential role of interleukin (IL)-11 and its receptor in breast cancer development and progression. However, there is little clinical information to support this hypothesis. We examined the expression of IL-11 and its receptor in primary breast cancer tissue samples and correlated their level of expression with the clinical outcome. METHODS: Primary breast cancer samples (n=109) and matched background tissue obtained from patients in the cohort (n=33) were processed for frozen section and RNA extraction. Frozen sections from matched tissues were immunostained with IL-11 and IL-11 receptor antibodies. Staining intensity was analyzed by computer image analysis. RNA was reverse-transcribed and quantified before analysis by quantitative polymerase chain reaction. Results were expressed as the number of transcripts (standardized by beta-actin). The data were compared with the clinical outcome of the disease. RESULTS: The intensity of staining for both IL-11 and the IL-11 receptor was distinctly high in tumor samples (P<.01). The transcript level of IL-11 was significantly higher in node-positive tumor samples compared with node-negative samples (P=.02). Tumors with a poor prognostic index and poor histological grade showed a higher level of IL-11. A higher level of IL-11 was linked to poorer survival with Kaplan-Meier survival analysis. CONCLUSIONS: IL-11 can be a predictor of poor prognosis in human breast cancer.

Breast Neoplasms↗
PubMed2005

Interlaboratory comparability study of cancer gene expression analysis using oligonucleotide microarrays.

A key step in bringing gene expression data into clinical practice is the conduct of large studies to confirm preliminary models. The performance of such confirmatory studies and the transition to clinical practice requires that microarray data from different laboratories are comparable and reproducible. We designed a study to assess the comparability of data from four laboratories that will conduct a larger microarray profiling confirmation project in lung adenocarcinomas. To test the feasibility of combining data across laboratories, frozen tumor tissues, cell line pellets, and purified RNA samples were analyzed at each of the four laboratories. Samples of each type and several subsamples from each tumor and each cell line were blinded before being distributed. The laboratories followed a common protocol for all steps of tissue processing, RNA extraction, and microarray analysis using Affymetrix Human Genome U133A arrays. High within-laboratory and between-laboratory correlations were observed on the purified RNA samples, the cell lines, and the frozen tumor tissues. Intraclass correlation within laboratories was only slightly stronger than between laboratories, and the intraclass correlation tended to be weakest for genes expressed at low levels and showing small variation. Finally, hierarchical cluster analysis revealed that the repeated samples clustered together regardless of the laboratory in which the experiments were done. The findings indicate that under properly controlled conditions it is feasible to perform complete tumor microarray analysis, from tissue processing to hybridization and scanning, at multiple independent laboratories for a single study.

Adenocarcinoma↗
PubMed1995

Virological characteristics of hepatitis C virus infection in chronic hemodialysis patients: a cross-sectional study.

Detection of hepatitis C virus viremia (HCV RNA) in serum of hemodialysis (HD) patients is crucial for documenting ongoing infection because the clinical and epidemiological importance of anti-HCV positivity is not clear. HCV viremia was studied in 104 HD patients by reverse transcription polymerase chain reaction (RT PCR) using primers localized in the 5' non-coding region of the viral genome. We used two different methods to detect HCV RNA: a direct PCR amplification of HCV RNA from human serum, and a standard RT PCR procedure (requiring the RNA extraction step). There were 50 (48%) anti-HCV positive patients in this population. Twenty-two (21.1%) out of 104 patients showed HCV RNA in serum by standard RT PCR technique: they belonged to the anti-HCV positive patient group, whereas all anti-HCV negative patients were HCV RNA negative. Prevalence of HCV RNA was more than doubled when standard RT PCR was used compared to direct RT PCR protocol. There was a good association between serum HCV RNA and circulating anti-HCV antibodies, tested by second-generation ELISA and RIBA assays. HCV viremia was not associated with either the presence or the absence of a particular RIBA antibody specificity. AST and ALT levels had no predictive value for HCV viremia, because they were repeatedly normal in the majority of viremic patients (16/22: 73%).(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-Sectional Studies↗