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Results for “Gene expression analysis”

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At least 343 records · Page 19Linked to original sources
PubMed2002

Expressed sequence tag-based gene expression analysis under aluminum stress in rye.

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.

Adaptation, Physiological↗
PubMed2003

Differential gene expression analysis in fish exposed to endocrine disrupting compounds.

This review discusses various methodologies that can be used to understand, at the gene level, the consequences to fish upon exposure to endocrine disrupting compounds (EDCs). Several approaches for measuring expression of gene transcripts are discussed, including directed approaches, such as Northern blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) as well as open-ended approaches, such as differential display RT-PCR, subtractive hybridizations, and gene arrays. Each of these systems has advantages and disadvantages, strengths and weaknesses. Conducting experiments with each of these methods provides important information about the molecular mechanisms that result from exposure to EDCs, information which can be used in risk assessment of polluted sites found in the environment.

Animals↗
PubMed2003

[Gene expression analysis in liver tissue at a single cell level by nested polymerase chain reaction and laser microdissection].

OBJECTIVE: To investigate the measurements of gene expressing at a single hepatocyte level. METHODS: Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR). RESULTS: Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells. CONCLUSION: Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.

Dissection↗
PubMed2004

A nucleic acid biosensor for gene expression analysis in nanograms of mRNA.

An ultrasensitive nucleic acid biosensor for direct detection of genes in mRNA extracted from animal tissues is described. It is based on amperometric detection of a target gene by forming an mRNA/redox polymer bilayer on a gold electrode. The mRNA was directly labeled with cisplatin-biotin conjugates through coordinative bonds with purine bases in the mRNA molecules. A subsequent binding of glucose oxidase-avidin conjugates to the labeled mRNA and the introduction of a poly(vinylimidazole-co-acrylamide) partially imidazole-complexed with [Os(bpy)(2)(im)] (bpy = 2,2'-bipyridine, im = imidazole) redox polymer overcoating to the electrode allowed for electrochemical detection of the oxidation current of glucose in solution. Depending on individual genes, detection limits of subfemtograms were achieved. As compared to a sandwich-type assay, the sensitivity was improved by as much as 25-fold through the incorporation of multiple enzyme labels to the mRNA molecules. Less than 2-fold gene expression difference was unambiguously differentiated in as little as 5.0 ng of mRNA. With the greatly improved sensitivity, at least 1000-fold more sensitive than fluorescence-based techniques, the amount of mRNA needed in the assay was cut down from microgram to nanogram levels.

Animals↗
PubMed2004

cDNA microarray gene expression analysis and toxicological phenotype for anticancer drug.

Toxicogenomics, the subdiscipline that merges genomics with toxicology, hold the promise to contributing toward the goal of elucidating mechanism by studying genomic profiling related with various drugs. The application of gene expression profiling technology to examine multiple genes and signaling pathways promises a significant advance in understanding the toxic mechanisms of various drugs and prediction of new drug candidate. Toxicogenomics is emerging field combining genomics and bioinformatics to identify and characterize mechanisms of toxicity of drug and various compounds. The principal hypothesis underlying on this field is that chemical-specific pattern of altered gene expression is related with each chemicals properties, especially toxicological property, and it will be revealed using high-density microarray analysis of sample from exposed organisms. So, in this study we compare the gene expression pattern of two anticancer drugs paclitaxel and orally absorbable paclitaxel, using the cDNA microarray. And from the result of this study, it is possible to provide the new possibility for genome-wide insight into mechanism of their anticancer activity and toxicological phenotype.

Administration, Oral↗
PubMed2006

The search for factors in human feeders that support the derivation and propagation of human embryonic stem cells: preliminary studies using transcriptome profiling by serial analysis of gene expression.

Serial analysis of gene expression (SAGE) was used to obtain the transcriptome profiles of a supportive human fetal skin feeder (Detroit 551) and a nonsupportive human fetal lung feeder (MRC-5) for human embryonic stem cells. A pairwise comparison of the two SAGE profiles showed that fibroblast growth factor-2 (FGF2), a bone morphogenetic protein 4 pathway inhibitor, Gremlin 1, and several extracellular matrix proteins that could potentially aid human embryonic stem cell attachment and growth were highly expressed in Detroit 551 fibroblasts.

Cell Culture Techniques↗
PubMed2004

Isolation and gene expression analysis of Arabidopsis thaliana mutants with constitutive expression of ATL2, an early elicitor-response RING-H2 zinc-finger gene.

Genes with unstable transcripts often encode proteins that play important regulatory roles. ATL2 is a member of a multigene family coding highly related RING-H2 zinc-finger proteins that may function as E3 ubiquitin ligases. ATL2 mRNA accumulation occurs rapidly and transiently after incubation with elicitors of pathogen response. We screened 50,000 M(2) families from a line that carries a fusion of pATL2 to the GUS reporter gene and isolated five mutants, which we named eca (expresión constitutiva de ATL2), that showed constitutive expression of the reporter gene. One mutant exhibits a drastic stunted phenotype while the other four grow similarly to wild type. Two early chitin-induced genes and known pathogenesis-related genes such as NPR1, PAL, and CHS are activated in all the mutants whereas members of the ATL family and PR-1 and PDF2.1, which are markers of the salicylic acid (SA) jasmonate (JA) defense-response pathways, display differential expression between the mutants. These observations indicate that the ECA gene products may function in the early steps of an elicitor-response pathway, although some of them may function at other stages on the SA or JA defense-response pathways. Likewise, the fact that ATL2 and other members of the ATL family are activated in eca mutants links the induction of this putative class of ubiquitin ligases to plant defense signaling pathways.

Arabidopsis↗
PubMed2002

[Technological advances of serial analysis of gene expression].

Serial analysis of gene expression (SAGE) is an effective method of determining gene expression profiles of tissues and organs under different conditions. In this paper, the detail protocol of SAGE was introduced and some modified procedure of SAGE was reviewed.

Gene Expression Profiling↗
PubMed2002

[Serial analysis of gene expression].

Serial analysis of gene expression (SAGE) is recently developed, a sequenced-based technique, which permits comprehensive and quantitative gene expression profiles from specific tissues or cells. SAGE has been successfully applied for transcriptome research and identification of differentially expressed genes between mRNA populations. This article mainly reviews the principle,development and application of SAGE.

English Abstract↗
PubMed2004

Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR.

To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment.

Animals↗
PubMed2002

The Burkholderia pseudomallei oxyR gene: expression analysis and mutant characterization.

Burkholderia pseudomallei (Bp) is the causative agent of the life-threatening melioidosis in humans. The global transcription factor oxyR gene was isolated and characterized. It is located between recG, encoding a putative DNA helicase, and katG, encoding a putative catalase-peroxidase. oxyR is expressed as a monocistronic 1 kb mRNA and is induced by oxidative stress compounds. Northern, primer extension, and transcription reporter fusion analyses showed that oxyR mRNA is induced by 0.2 mM menadione, 2 mM paraquat, and 10 mM H(2)O(2). Two knockout mutants of oxyR were constructed, by single- and double-crossover recombination, and found to be hypersensitive to H(2)O(2) and paraquat. Bp lacking OxyR exhibited autoaggregation when cultured in liquid broth and an increased ability to form biofilms in minimal medium, but not in Luria-Bertani broth. The oxyR mutants also have a decreased level of extracellular protease activity. The altered phenotypes of oxyR deficient mutants were complemented when a copy of oxyR was transposed into the mutant chromosomes on the mini-Tn5 transposon.

Bacterial Proteins↗
PubMed2003

Gene expression analysis in interstitial lung edema induced by saline infusion.

To investigate the molecular events taking place during the development of hydraulic interstitial edema, we analyzed by microarray and conventional molecular techniques the variation of gene expression in lung from rabbits treated with slow-rate saline infusions. This analysis indicates that even a condition characterized by a small increase in extravascular water can have a significant influence on the inflammatory milieu. In this regard, cytokines, in particular TNFalpha, can be considered early mediators capable of inducing secondary effects on the injured tissue. Moreover, two MT1 genes were strongly up-regulated, data consistent with their role as protective molecules.

Animals↗
PubMed2006

Gene expression analysis indicates extensive genotype-specific crosstalk between the conjugative F-plasmid and the E. coli chromosome.

BACKGROUND: Plasmids are an important component of the bacterial genome, but the crosstalk between genes encoded on the chromosome and on the plasmid is still poorly understood. RESULTS: We performed a large-scale survey for genes on the E. coli chromosome that are affected by the presence of the conjugative F-plasmid (crosstalk). The expression pattern of about 4% (107 genes) of the genes encoded by the chromosome was affected by the presence of the F-plasmid. Comparing two different Escherichia coli strains, MG1655 and DH5alpha, we found a strong host genotype-specific crosstalk of the host chromosome with the F-plasmid. About 88% of the genes affected by the presence of the F-plasmid showed a significant plasmid by host genotype interaction, i.e. the presence of the F-plasmid resulted in a different gene expression in the two host genotypes. Less than 12% of the genes showed an additive effect of gene expression, i.e. host genotype independent crosstalk between plasmid and host chromosome. CONCLUSION: We propose that epistatic effects also contribute to the maintenance of F-plasmids in natural populations.

Analysis of Variance↗
PubMed2003

Gene expression analysis of atopic dermatitis-like skin lesions induced in NC/Nga mice by mite antigen stimulation under specific pathogen-free conditions.

BACKGROUND: Atopic dermatitis (AD) is a chronic relapsing inflammation characterized by pruritic and eczematous skin lesions usually observed in patients with a familial history of atopic diseases, but its exact etiology is unclear. An animal model is indispensable for the analysis of the pathogenesis and the development of new drugs to treat this disease. Here, we compare changes in gene expression profiles in the AD-like skin lesions of NC/Nga or BALB/c mice stimulated intradermally by mite antigen under specific pathogen-free (SPF) conditions. METHODS: Mite Extract-Dp was injected intradermally into the right and left pinnae and into the skin of the back of NC/Nga or BALB/c mice in 2 places once per 3 days, and the clinical symptoms and the ear thickness were measured. On day 14 or day 28 after starting mite extract injection, we collected plasma and pinnae from NC/Nga or BALB/c mice. The amount of total immunoglobulin E (IgE) in plasma was assayed. We analyzed mRNA transcripts in pinnae using real-time quantitative PCR for the murine counterparts of several known allergy-related genes. Moreover, genome-wide gene expression in pinnae from NC/Nga mice was analyzed using high-density oligonucleotide arrays (GeneChip, Affymetrix). RESULTS: From 2 weeks after stimulation, marked skin inflammation was induced in the pinnae of NC/Nga but not BALB/c mice. However, IgE levels in sera rose equally in both strains. Quantitative PCR analysis and comprehensive GeneChip analysis of the AD-like pinna skin lesions revealed that their development was accompanied by changes in expression of more than 1,000 genes. These included cytokines, cytokine receptors, proteases, and adhesion molecules. Furthermore, genes thus far not reported in association with AD were also affected. CONCLUSION: From these results, the NC/Nga mouse model using mite sensitization under SPF conditions could be useful for elucidating the mechanisms of AD pathogenesis and developing more effective therapy for AD.

Animals↗
PubMed2001

Determining significant fold differences in gene expression analysis.

A typical use for RNA expression microarrays is comparing the measurement of gene expression of two groups. There has not been a study reproducing an entire experiment and modeling the distribution of reproducibility of fold differences. Our goal was to create a model of significance for fold differences, then maximize the number of ESTs above that threshold. Multiple strategies were tested to filter out those ESTs contributing to noise, thus decreasing the requirements of what was needed for significance. We found that even though RNA expression levels appears consistent in duplicate measurements, when entire experiments are duplicated, the calculated fold differences are not as consistent. Thus, it is critically important to repeat as many data points as possible, to ensure that genes and ESTs labeled as significant are truly so. We were successfully able to use duplicated expression measurements to model the duplicated fold differences, and to calculate the levels of fold difference needed to reach significance. This approach can be applied to many other experiments to ascertain significance without a priori assumptions.

Expressed Sequence Tags↗
PubMed2005

mRNA and 18S-RNA coapplication-reverse transcription for quantitative gene expression analysis.

Fluorescence-based reverse transcription real-time quantitative polymerase chain reaction (RT-QPCR) is a highly sensitive method for the detection and quantitation of mRNA. To control and correct for sample variability, some common housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and ubiquitin are often used as endogenous standards. Other internal calibrators such as 18S-ribosomal RNA (18S-RNA) have also been used, but further methodological concerns arise given that ribosomal RNA lacks the 3' poly-A tail typically associated with messenger RNA. To take advantage of the constant expression levels of 18S-RNA and the precision of oligo-(dT) primed first-strand synthesis, we have developed a method that combines oligo-(dT) with an 18S-RNA-specific primer in the initial reverse transcription (RT) reaction. This strategy, termed coapplication reverse transcription (Co-RT), allows for the analysis of multiple target genes with the advantages of 18S-RNA normalization from a single RT reaction. In this article, we describe Co-RT and present tissue distribution and expression level analysis of several target genes using this method. Co-RT provides increased sensitivity and higher accuracy than do the standard random primed RT methods.

Animals↗
PubMed2005

Gene expression analysis of cultured amniotic fluid cell with Down syndrome by DNA microarray.

Complete or partial triplication of human chromosome 21 results in Down syndrome (DS). To analyze differential gene expressions in amniotic fluid (AF) cells of DS, we used a DNA microarray system to analyze 102 genes, which included 24 genes on chromosome 21, 28 genes related to the function of brain and muscle, 36 genes related to apoptosis, 4 genes related to extracellular matrix, 8 genes related to other molecular function and 2 house-keeping genes. AF cells were collected from 12 pregnancies at 16-18 weeks of gestation in DS (n=6) and normal (n=6) subjects. Our DNA microarray experiments showed that the expressions of 11 genes were altered by at least 2-folds in DS, as follows. Ten genes, COL6A1, CASP5, AKT2, JUN, PYGM, BNIP1, OSF-2, PRSS7, COL3A1, and MBLL were down-regulated and GSTT1 was only up-regulated. The differential expressions of GSTT1 and COL3A1 were further confirmed by semi-quantitative RT-PCR for each sample. The gene dosage hypothesis on chromosome 21 may explain the neurological and other symptoms of DS. However, our results showed that only two genes (COL6A1 and PRSS7), among 24 genes on chromosome 21, were down-regulated in the AF cells of DS. Our data may provide the basis for a more systematic identification of biological markers of fetal DS, thus leading to an improved understanding of pathogenesis for fetal DS.

Amniocentesis↗