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Results for “Gene expression analysis”

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At least 595 records · Page 33Linked to original sources
PubMed2004

Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes.

The accuracy of 18S rRNA, beta-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, beta-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of beta-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gammadeltaTCR(+), CD4(+) and CD8(+) subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30-70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.

Actins↗
PubMed2006

Abundant transcripts of malting barley identified by serial analysis of gene expression (SAGE).

Serial analysis of gene expression (SAGE) was applied to the major cereal crop barley (Hordeum vulgare) to characterize the transcriptional profile of grain during the malting process. Seven SAGE libraries were generated from seed at different time points during malting, in addition to one library from dry mature seed. A total of 155,206 LongSAGE tags, representing 41,909 unique sequences, was generated. This study reports an in-depth analysis of the most abundant transcripts from each of eight specific time points in a malting barley time course. The 100 most abundant tags from each library were analysed to identify the putative functional role of highly abundant transcripts. The largest functional groups included transcripts coding for stress response and cell defence, ribosomal proteins and storage proteins. The most abundant tag represented B22EL8, a barley metallothionein, which showed significant up-regulation across the malting time course. Considerable changes in the abundance profiles of some of the highly abundant tags occurred at 24 h post-steeping, indicating that it may be an important time point for gene expression changes associated with barley seed germination.

Base Sequence↗
PubMed1999

Analysis of gene expression in single cells.

A cell's structural and functional characteristics are dependent on the specific complement of genes it expresses. The ability to study and compare gene usage at the cellular level will therefore provide valuable insights into cell physiology. Such analyses are complicated by problems associated with sample collection, sample size and the limited sensitivity of expression assays. Advances have been made in approaches to the collection of cellular material and the performance of single-cell gene expression analysis. Recent development in global amplification of mRNA may soon permit expression analyses of single cells to be performed on DNA microarrays.

Gene Expression↗
PubMed2003

Electrophoretic signal comparison applied to mRNA differential display analysis.

Gene expression analysis by electrophoretic methods is currently limited by the labor-intensive visual evaluation of the electrophoretic signal profiles. For this purpose, we present a flexible approach to computer-assisted comparison of quantitative electrophoretic patterns between multiple expression signals. Gaussian curves are first fitted to the complex peak mixtures, and the resulting approximate signals are then aligned and compared on a peak-by-peak basis with respect to specific patterns defined by the investigator. The rationale of the method is to produce a compressed list of exceptional expression patterns quantified by a set of associated numeric features. A score value is attached to each pattern in such a way that large values identify the most potential findings to be focused on in visual analysis instead of the vast amount of original electrophoretic results. The validity of the method is demonstrated by analyzing a large set of electrophoretic data from mRNA differential display experiments monitoring changes in gene expression patterns in human colonic carcinoma. The automated identification of variously defined gene expression patterns agrees well with the visual evaluation of the same electropherograms. The general comparison approach may also be found useful with other gene expression profiling instruments.

Adenocarcinoma↗
PubMed2006

Analysis of gene expression during development: lessons from the Apicomplexa.

Apicomplexans are responsible for significant human and animal disease worldwide, including malaria and toxoplasmosis. Herein we summarize recent advances in gene expression analysis in these eukaryotic pathogens, especially with respect to their developmental biology, and discuss the impact this work may have on the development of new vaccines and chemotherapeutics.

Animals↗
PubMed2002

Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice.

Macrophage foam cells are integral in the development of atherosclerotic lesions. Gene expression analysis of lesional macrophage foam cells is complicated by the cellular heterogeneity of atherosclerotic plaque and the presence of lesions of various degrees of severity. To overcome these limitations, we tested the ability of laser capture microdissection (LCM) and real-time quantitative reverse transcription PCR to selectively analyze RNA from lesional macrophages of apolipoprotein E (apoE)-deficient mice. Proximal aortic tissue sections were immunostained for macrophagespecific CD68/macrosialin by a rapid (approximately 15-min) protocol. Alternating sections from each animal were used to isolate RNA either from entire sections (analogous to isolation from whole tissue) or by LCM selection of CD68-positive cells. We measured the mRNA levels of CD68, a macrophage-specific marker, alpha-actin, a smooth muscle cell marker, and cyclophilin A, a control gene. Compared with whole sections, CD68 mRNA levels were greatly enriched (33.6-fold) in the laser-captured lesional macrophages. In contrast to whole sections, LCM-derived RNA had undetectable levels of alpha-actin. To illustrate the ability of this method to measure changes in lesional macrophage gene expression, we injected 100 microg of lipopolysaccharide i.p. into apoE-deficient mice and detected in laser-captured lesional macrophages increased mRNA expression for vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, and monocyte chemoattractant protein-1 (11.9-, 32.5-, and 31.0-fold, respectively). By selectively enriching foam cell RNA, LCM provides a powerful approach to study the in situ expression and regulation of atherosclerosis-related genes. This approach will allow the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.

Actins↗
PubMedDate not supplied

[A new method of comparative analysis of gene expression and identification of differentially expressed mRNA].

A novel method of comparative gene expression analysis is proposed which is based on representing a population of expressed mRNA sequences in the form of a set of discrete cDNA restriction fragments. By means of selective isolation of 3' terminal cDNA fragments, each mRNA species is represented by no more than one cDNA fragment of specific length and sequence. Populations of cDNA fragments from different cell types are separated by high-resolution gel electrophoresis, and the separation patterns are compared. Using the proposed approach, fragments of two genes differentially expressed in murine thymus and spleen were identified and cloned. One of the genes was found to encode the terminal deoxynucleotidyltransferase, the there is apparently a heretofore unknown gene.

Animals↗
PubMed1998

Analysis of gene expression in osteogenic cultured marrow/hydroxyapatite construct implanted at ectopic sites: a comparison with the osteogenic ability of cancellous bone.

We investigated the in vivo osteogenic ability of cultured marrow cells subcultured in porous hydroxyapatite. This osteogenic ability was compared with that of cancellous bone grafts. Fresh marrow cells were obtained from young adult rat femora and cultured in a standard medium for 10 days, then trypsinized and used to make constructs of porous hydroxyapatite (HA) and cultured marrow cells. An additional 2-week culture (subculture) was performed for the construct in standard medium with and without the addition of dexamethasone (Dex). The 2-week subcultured constructs then were implanted into subcutaneous sites of syngeneic rats. These implants and the rat cancellous bone were harvested and prepared for gene expression analysis of alkaline phosphatase (ALP) and osteocalcin (OC) as well as for histological analysis. ALP and OC mRNAs could be detected in Dex-treated subcultured constructs 1 week after implantation with an increase at 2 weeks, comparable to the observation in cancellous bone. Histological analysis showed active bone formation even 1 week postimplantation. In contrast, the subcultured constructs without the addition of Dex did not show bone formation, and only small levels of ALP and OC mRNAs were found. These results indicate that the bone tissue formed by grafting the Dex-treated construct of cultured marrow cells and hydroxyapatite possesses a high osteoblastic activity comparable to that of viable cancellous bone. Thus the prefabricated osteogenic subcultured marrow/HA construct may be applicable in bone reconstructive surgery.

Alkaline Phosphatase↗
PubMed2006

Transcriptome analyses of male germ cells with serial analysis of gene expression (SAGE).

Serial analysis of gene expression (SAGE) provides an alternative with additional advantages to microarrays for studying gene expression during spermatogenesis. The digitized transcriptome provided by SAGE of purified mouse germ cells identified 27,504 species of transcripts expressed in type A spermatogonia, pachytene spermatocytes, and round spermatids. Over 2700 of these transcripts were novel. Computational analyses allowed the identification of clusters of co-regulated genes, cell-specific promoter modules, cell-specific biological processes, as well as "preferential" biological networks in different cell types. These analyses provided potential drug targets for interference of specific pathways at different stages of spermatogenesis. Analyses of the transcriptomes revealed the prominent role of cytochrome c oxidase in germ cells and suggest a novel role for this enzyme in cytochrome c-mediated apoptosis in spermatogonia. A number of genes were shown to undergo differential splicing during spermatogenesis giving rise to cell-specific splice variants.

Alternative Splicing↗
PubMed2003

Serial analysis of gene expression (SAGE): advances, analysis and applications to pigment cell research.

As cells progress from normal to diseased states, they may undergo a series of gene expression changes. Advances in molecular biology allow us to examine a host of these changes at once, in a high throughput fashion. Serial analysis of gene expression (SAGE) allows for the expression profiling of the complete transcriptome of a given cell, and has the potential for identifying novel genes as well as those in low abundance. In this review, we will outline the technique, how one analyzes the massive amounts of data generated, and describe pigment cell libraries currently in the making.

Gene Expression Profiling↗
PubMed2003

Serial analysis of gene expression and cancer.

Serial analysis of gene expression has been widely used to characterize gene expression patterns associated with tumor formation. These studies resulted in the identification of tumor-specific markers, transcriptional pathways, or therapeutic targets. In this review, recent applications and developments of serial analysis of gene expression and their impact on the diagnosis and treatment of cancer are discussed. A combination of serial analysis of gene expression and small-scale microarray analysis represents a strategy that should facilitate the identification and exploitation of tumor-specific gene expression for diagnostic or therapeutic purposes. In addition, cancer diagnosis and treatment may benefit from a complementation between serial analysis of gene expression and quantitative proteomics in the future.

Biomarkers, Tumor↗
PubMed2004

A Serial Analysis of Gene Expression (SAGE) database analysis of chemosensitivity: comparing solid tumors with cell lines and comparing solid tumors from different tissue origins.

Drug sensitivity and resistance has been most extensively studied in cell lines carried in tissue culture. Furthermore, cell lines have been widely used in testing new anticancer agents, despite the widely recognized observation that cell lines are more sensitive to cytotoxic drugs than are their corresponding solid tumors. We used the Serial Analysis of Gene Expression (SAGE) database to identify differences between solid tumors and cell lines, hoping to detect genes that could potentially explain differences in drug sensitivity. SAGE libraries were available for both solid tumors and cell lines from breast, colon, ovarian, pancreatic, and prostate carcinomas and from gliomas and medulloblastomas. Sixty-two genes were identified as overexpressed in tumors. The immune response and complement pathways were the significant common themes, with extracellular matrix (ECM) proteins third. For the 61 genes overexpressed in cell lines, protein synthesis was the dominant theme. We next used the SAGE database to identify genetic differences between tumor types that convey a broad range of survival to the patients that bear them as distant metastases. SAGE gene expression data were correlated with 5-year survivals documented in the SEER (Surveillance, Epidemiology and End-Results) database for patients diagnosed with "distant" or metastatic cancers. These are unlikely to be amenable to surgical resection; therefore, survival here reflects, to some extent, sensitivity to systemic therapy, i.e., chemotherapy. Using survival data as a surrogate of chemotherapy sensitivity, a spectrum can be generated, with testicular cancer at one end and pancreatic cancer at the other. Favorable 5-year survival, despite a distant presentation, correlates with expression of protein synthesis genes. Poor 5-year survival correlates with expression of cell adhesion, cytoskeletal, and ECM genes, a pattern similar to that found to distinguish solid tumors from the more cytotoxin-sensitive cancer cell lines. One interpretation is that resistance to chemotherapy may arise, in part, from the adherent, relatively inert condition (i.e., low in protein synthesis potential) of refractory cancers. Thus, attachment or ECM genes could be targets for anticancer therapy.

Cell Line, Tumor↗
PubMed2001

Identification of androgen-regulated genes in the prostate cancer cell line LNCaP by serial analysis of gene expression and proteomic analysis.

A common therapy for nonorgan-confined prostate cancer involves androgen deprivation. To develop a better understanding of the effect of androgen on prostatic cells, we have analyzed gene expression changes induced by dihydrotestosterone (DHT) in the androgen responsive prostate cancer line LNCaP, at both RNA and protein levels. Changes at the RNA level induced by DHT were determined by means of serial analysis of gene expression (SAGE), and protein profiling was done by means of quantitative two-dimensional polyacrylamide gel electrophoresis. Among 123,371 transcripts analyzed, a total of 28,844 distinct SAGE tags were identified representing 16,570 genes. Some 351 genes were significantly affected by DHT treatment at the RNA level (p < 0.05), of which 147 were induced and 204 repressed by androgen. In two independent experiments, the integrated intensity of 32 protein spots increased and 12 decreased at least two-fold in response to androgen, out of a total of 1031 protein spots analyzed. The change in intensity for most of the affected proteins identified could not be predicted based on the level of their corresponding RNA. Our study provides a global assessment of genes regulated by DHT and suggests a need for profiling at both RNA and protein levels for a comprehensive evaluation of patterns of gene expression.

Androgens↗
PubMed2004

Resolution of large and small differences in gene expression using models for the Bayesian analysis of gene expression levels and spotted DNA microarrays.

BACKGROUND: The detection of small yet statistically significant differences in gene expression in spotted DNA microarray studies is an ongoing challenge. Meeting this challenge requires careful examination of the performance of a range of statistical models, as well as empirical examination of the effect of replication on the power to resolve these differences. RESULTS: New models are derived and software is developed for the analysis of microarray ratio data. These models incorporate multiplicative small error terms, and error standard deviations that are proportional to expression level. The fastest and most powerful method incorporates additive small error terms and error standard deviations proportional to expression level. Data from four studies are profiled for the degree to which they reveal statistically significant differences in gene expression. The gene expression level at which there is an empirical 50% probability of a significant call is presented as a summary statistic for the power to detect small differences in gene expression. CONCLUSIONS: Understanding the resolution of difference in gene expression that is detectable as significant is a vital component of experimental design and evaluation. These small differences in gene expression level are readily detected with a Bayesian analysis of gene expression level that has additive error terms and constrains samples to have a common error coefficient of variation. The power to detect small differences in a study may then be determined by logistic regression.

Algorithms↗
PubMed2005

Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of PrP gene expression and genetic susceptibility to scrapie.

BACKGROUND: Cellular prion protein expression is essential for the development of transmissible spongiform encephalopathies (TSEs), and in sheep, genetic susceptibility to scrapie has been associated to PrP gene polymorphisms. To test the hypothetical linkage between PrP gene expression and genetic susceptibility, PrP mRNA levels were measured by real-time RT-PCR in six ovine tissues of animals with different genotypes. RESULTS: Previous to the PrP gene expression analysis the stability of several housekeeping (HK) genes was assessed in order to select the best ones for relative quantification. The normalisation of gene expression was carried out using a minimum of three HK genes in order to detect small expression differences more accurately than using a single control gene. The expression stability analysis of six HK genes showed a large tissue-associated variation reflecting the existence of tissue-specific factors. Thereby, a specific set of HK genes was required for an accurate normalisation of the PrP gene expression within each tissue. Statistical differences in the normalised PrP mRNA levels were found among the tissues, obtaining the highest expression level in obex, followed by ileum, lymph node, spleen, cerebellum and cerebrum. A tendency towards increased PrP mRNA levels and genetic susceptibility was observed in central nervous system. However, the results did not support the hypothesis that PrP mRNA levels vary between genotypes. CONCLUSION: The results on PrP gene expression presented here provide valuable baseline data for future studies on scrapie pathogenesis. On the other hand, the results on stability data of several HK genes reported in this study could prove very useful in other gene expression studies carried out in these relevant ovine tissues.

Journal Article↗
PubMed1999

Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources.

We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE). Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source. A major fraction (>20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of approximately 6300 yeast genes. Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters). In oleate-grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal beta-oxidation enzymes required for degradation of fatty acids. The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes. We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2 and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins. Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment. We describe a statistical method for analysis of data obtained by SAGE.

Carbon↗
PubMed2001

Oligonucleotide microarray expression analysis of genes whose expression is correlated with tumorigenic and non-tumorigenic phenotype of HeLa x human fibroblast hybrid cells.

In order to understand the differences and similarities between tumorigenic and non-tumorigenic HeLaxhuman fibroblast hybrids, gene expression profiles were examined with synthetic oligonucleotide arrays containing nearly 7000 gene probe sets. We used two pairs of genetically related hybrids, each pair representing individual clones of non-tumorigenic and tumorigenic segregant hybrids, respectively. Analysis of six possible comparisons, utilizing two algorithms, identified 204 genes with differential expression. The greater number of differentially expressed genes was observed when non-tumorigenic hybrids were compared with tumorigenic segregants. Fifteen and 14 genes, respectively, were consistently found to be differentially expressed in non-tumorigenic and tumorigenic cells. Among those 29 differentially expressed genes, three (intestinal alkaline phosphatase, caveolin-1, and solute carrier family2, member3) have been reported previously to be associated with the tumorigenic phenotype, using the same hybrid pairs. In addition, among the genes previously detected by differential display, 78% of them exhibited more than 5-fold change, demonstrating a high consistency between the two methods of differential gene expression. These findings suggest that synthetic oligonucleotide arrays are a powerful and highly reproducible tool to identify those genes whose expression is associated with certain phenotypes.

Algorithms↗
PubMed2006

Databases of free expression.

The rapid development of microarray technologies has led to a similar progression in gene expression analysis methods, gene expression applications, and gene expression databases. Public gene expression databases enable any researcher to examine expression of their favorite genes across a wide variety of samples, download sample data for development of new analysis methods, or answer broad questions about gene expression regulation, among other applications. A wide variety of public gene expression databases exist, and they vary in their content, analysis capabilities, and ease of use. This review highlights the current features and describes examples of two broad categories of mammalian microarray databases: tissue gene expression databases and data warehouses.

Animals↗