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At least 199 records · Page 11Linked to original sources

PubMed2004

Gene expression profile in corneal neovascularization identified by immunology related macroarray.

PURPOSE: To identify differentially expressed genes in corneal neovascularization using cDNA macroarray. METHODS: Mechanical denudation of corneal and limbal epithelium was performed to induce corneal vascularization in mice. Corneas were harvested 4 days after operation. Total RNA was isolated from both normal and vascularized corneas and used for the synthesis of cDNA probes. 32P labeled exponential cDNA probes were hybridized to mouse cDNA immunology arrays. To validate the gene expression patterns revealed by the cDNA expression array analysis, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and statistical analysis were performed to compare the normal and vascularized corneal samples. RESULTS: Of 545 immunology related genes on the arrays, 6 genes were upregulated and 1 gene was downregulated in the vascularized corneas compared with the normal corneas. Semi-quantitative RT-PCR was performed on the 6 genes selected in the arrays study, and showed that calreticulin (calregulin), apolipoprotein E, HSP84 (HSP90beta), and pleiotrophin were upregulated while interferon regulatory factor-1 was downregulated in the vascularized corneas compared with the normal corneas. These genes possess a number of biological functions including molecular chaperon, growth factor, and transcriptional factors. CONCLUSIONS: The differentially expressed genes newly identified in the context of corneal neovascularization represent novel candidate factors for further functional studies of the mechanisms of corneal neovascularization. Our data may provide new insight into the biological process of inflammation induced corneal neovascularization.

Animals↗

PubMed2002

The limit fold change model: a practical approach for selecting differentially expressed genes from microarray data.

BACKGROUND: The biomedical community is developing new methods of data analysis to more efficiently process the massive data sets produced by microarray experiments. Systematic and global mathematical approaches that can be readily applied to a large number of experimental designs become fundamental to correctly handle the otherwise overwhelming data sets. RESULTS: The gene selection model presented herein is based on the observation that: (1) variance of gene expression is a function of absolute expression; (2) one can model this relationship in order to set an appropriate lower fold change limit of significance; and (3) this relationship defines a function that can be used to select differentially expressed genes. The model first evaluates fold change (FC) across the entire range of absolute expression levels for any number of experimental conditions. Genes are systematically binned, and those genes within the top X% of highest FCs for each bin are evaluated both with and without the use of replicates. A function is fitted through the top X% of each bin, thereby defining a limit fold change. All genes selected by the 5% FC model lie above measurement variability using a within standard deviation (SDwithin) confidence level of 99.9%. Real time-PCR (RT-PCR) analysis demonstrated 85.7% concordance with microarray data selected by the limit function. CONCLUSION: The FC model can confidently select differentially expressed genes as corroborated by variance data and RT-PCR. The simplicity of the overall process permits selecting model limits that best describe experimental data by extracting information on gene expression patterns across the range of expression levels. Genes selected by this process can be consistently compared between experiments and enables the user to globally extract information with a high degree of confidence.

Animals↗

PubMed2002

Kinetics of gene expression profiling in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke.

Previous studies from different laboratories have demonstrated that cigarette smoke (CS) harbours a strong oxidative stress potential, which broadly impacts exposed cells. Many of these studies have been devoted to identifying differentially expressed genes in exposed cells. Emerging DNA microarray techniques provide a sophisticated tool to characterize gene expression on a more comprehensive basis. Here, we report on kinetic studies performed to characterize gene expression profiles in Swiss 3T3 cells exposed to aqueous extracts of CS ('smoke-bubbled phosphate-buffered saline') up to 24 h through glass chips containing 513 different cDNA probes. The results obtained display a distinct expression pattern of up regulated and repressed genes, which was most evident after 4-8 h of exposure. The CS-related stress response involves mainly antioxidant response genes coding for, e.g. haem oxygenase-1 (HO-1), metallothionein 1/2 (MT1/2) and heat shock proteins (HSPs); genes coding for transcription factors, e.g. JunB and CAAT/enhancer binding protein (C/EBP); cell cycle-related genes, e.g. gadd34 and gadd45; and notably, genes described as mediators of an inflammatory/immune-regulatory response, e.g. st2, kc and id3. From a kinetic perspective, the stress response is characterized by the synchronized up regulation of antioxidant pathways, e.g. as reflected by the co-ordinated expression of ho-1 and ferritin. This expression pattern is obviously orchestrated by stress-responsive transcription factors, as exemplified by the early and strong expression of junB and c/ebp. Interestingly, among the 10 most up regulated genes are five which are known to counteract stress brought about by peroxynitrite. Altogether, these results demonstrate that CS induces a distinct signature of differential gene expression in exposed cells.

3T3 Cells↗

PubMed2003

Growth hormone down-regulation of Interleukin-1beta and Interleukin-6 induced acute phase protein gene expression is associated with increased gene expression of suppressor of cytokine signal-3.

Severe burn induces the hepatic acute phase response. We previously showed that recombinant human growth hormone (GH) treatment after burn down-regulated acute phase protein (APP) production and gene expression in vivo. In this study, we hypothesized that the inhibitory effect of GH on the hepatic acute phase response was due to increased suppressor of cytokine signaling (SOCS) gene expression. HepG2 cells were treated with Interleukin-1beta (IL-1beta; 2 ng/mL) and interleukin 6 (IL-6; 20 ng/mL) alone or combined with GH (2 microg/mL) for 15 and 30 min, and 1, 2, and 4 h. The levels of gene expression for alpha1-acid glycoprotein (AGP), alpha1-antitrypsin (ATT), and SOCS (CIS, SOCS-1, 2, and 3) were measured by reverse transcript-polymerase chain reaction (RT-PCR). APP levels in the supernatant were determined by enzyme-linked immunosorbent sandwich assay (ELISA). The gene expression of AGP and ATT were also measured in HepG2 cells transfected with pEF-Flag-l/mSOCS-3 plasmid after IL-1beta or IL-6 treatment. Data are expressed as means +/- SEM, and statistical analysis was performed by one- or two-way analysis of variance. IL-1beta and IL-6 induced AGP and ATT gene expression and protein production, respectively, which was down-regulated by GH treatment. SOCS-3 but not CIS, SOCS-1, or SOCS-2 gene expression was significantly increased by GH treatment. APP gene expression was significantly decreased in cells transfected with plasmid over expressing SOCS-3 after IL-6 and IL-1beta treatment. GH attenuates IL-1beta or IL-6 induced APP gene expression, which is associated with increased expression of SOCS-3. This study suggests that SOCS-3 plays an important role in the suppression of cytokine signaling by GH in down-regulating the acute phase response after injury.

Acute-Phase Proteins↗

PubMedDate not supplied

Raloxifene covalently bonded to titanium implants by interfacing with (3-aminopropyl)-triethoxysilane affects osteoblast-like cell gene expression.

Since Raloxifene, a drug used in osteoporosis therapy, inhibits the osteoclast functions but not osteoblast functions, it could improve the recovery during implant surgery. This preliminary report describes a simple method to link, through a covalent bond, Raloxifene to titanium by interfacing with (3-aminopropyl)-Triethoxysilane as assessed by the IR-FT and SEM. To evaluate the biological response of osteoblast-like cells to this implant, we compared expression gene profiling of cell cultures on Raloxifene conjugated implant and normal implant by DNA microarray. By using DNA microarrays containing 19,200 genes, we identified differently expressed genes in osteoblast-like cell line (MG-63). Surface Raloxifene conjugated implants have been shown to have a relevant importance in modifying cell response. This result could be an interesting starting point for the use of an immediate functional loading of implants.

Cell Line↗

PubMed2006

Robust gene expression with amplified RNA from biopsy-sized human heart tissue.

The need to assess heart failure at an early stage highlights the importance of accurate microarray analysis using small tissue samples. To test our ability to obtain high quality RNA from biopsy-sized cardiac specimens, amplification was performed on RNA from biopsy-sized samples of left ventricle (LV) tissue from one explanted failing human heart and one non-failing heart. Two methods were used: one-cycle (1C) amplification of 1.6 microg of RNA, and two-cycle (2C) amplification of 50 ng of RNA. The resulting cRNA was hybridized to Affymetrix GeneChip arrays. Over 65% of all differentially expressed genes for failing vs non-failing hearts were concordant between 1C and 2C RNA amplification. Differentially expressed genes between 1C and 2C RNA amplification in our study were highly correlated (R(2) = 0.957 and changes in gene expression agreed with prior studies on genes and heart failure; e.g., decreased alpha-myosin heavy chain and alpha-tropomyosin, as well as increased expression of insulin-like growth factor). Two cycles of amplification from cardiac biopsies will permit accurate transcription profiling of heart failure at pre-symptomatic stages. Ability to measure gene expression from nanogram amounts of RNA will provide new opportunities to predict progression to symptomatic heart failure, and to identify potential targets for therapy.

Biopsy↗

PubMed2001

Combination of PCR subtraction and cDNA microarray for differential gene expression profiling.

PCR subtraction hybridization has been used effectively to enrich and single out differentially expressed genes. However identification of these genes by means of cloning and sequencing individual cDNAs is a tedious and lengthy process. In this report, an attempt has been made to combine the use of PCR select cDNA subtraction hybridization and cDNA microarrays to identify differentially expressed genes using a nonradioactive chemiluminescent detection method. mRNA from human prolactin (hPRL) or human prolactin antagonist (hPRL-G129R) treated and non-treated breast cancer cells was isolated, and cDNAs were synthesized and used for the PCR subtraction to enrich the differentially expressed genes in the treated cells. The PCR-amplified and subtracted cDNA pools were purified and labeled using the digoxigenin method. Labeled cDNAs were hybridized to a human apoptosis cDNA microarray membrane and identified by chemiluminescence. The results suggest that the strategy of combining all three methods will allow for a more efficient, nonradioactive way of identifying differentially expressed genes in target cells.

Biotechnology↗

PubMed2000

cDNA-RDA of genes expressed in fetal and adult lungs identifies factors important in development and function.

The identification of genetic factors important in lung development and function will help in understanding the underlying molecular mechanisms of respiratory disease. Representational difference analysis of cDNA (cDNA-RDA) is a PCR-based subtractive enrichment procedure for the isolation of differentially expressed genes. We performed cDNA-RDA and isolated genes expressed more abundantly in fetal and adult lungs. Fifty-four clones potentially representing genes with higher transcript levels in the fetal lung were sequenced. Sequence similarity searches indicated that these clones included 12 known genes, a discoidin-like domain-containing gene, six expressed sequence tags (ESTs), and one novel sequence. Fifty-six clones potentially representing genes expressed more abundantly in the adult lung were also cloned and sequenced. Of these, 16 known human genes were represented along with two sequences significantly similar to known mouse genes and two novel sequences. Several of these known genes are implicated in stress response and lung protection. Thus cDNA-RDA was successfully used to isolate known and novel differentially expressed genes, which putatively play an important role in human lung development.

Adolescent↗

PubMed2000

Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification.

We have developed a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 10 bases into their corresponding 3' cDNA fragments covering hundred bases. A primer containing the 10-base SAGE tag is used as the sense primer, and a single base anchored oligo(dT) primer is used as an antisense primer in PCR, together with Pfu DNA polymerase. By using this approach, a cDNA fragment extending from the SAGE tag toward the 3' end of the corresponding sequence can be generated. Application of the GLGI technique can solve two critical issues in applying the SAGE technique: one is that a longer fragment corresponding to a SAGE tag, which has no match in databases, can be generated for further studies; the other is that the specific fragment corresponding to a SAGE tag can be identified from multiple sequences that match the same SAGE tag. The development of the GLGI method provides several potential applications. First, it provides a strategy for even wider application of the SAGE technique for quantitative analysis of global gene expression. Second, a combined application of SAGE/GLGI can be used to complete the catalogue of the expressed genes in human and in other eukaryotic species. Third, it can be used to identify the 3' cDNA sequence from any exon within a gene. It can also be used to confirm the reality of exons predicted by bioinformatic tools in genomic sequences. Fourth, a combined application of SAGE/GLGI can be applied to define the 3' boundary of expressed genes in the genomic sequences in human and in other eukaryotic genomes.

Cloning, Molecular↗

PubMed2000

Genome changes and gene expression in human solid tumors.

Genome-wide analysis techniques such as chromosome painting, comparative genomic hybridization, representational difference analysis, restriction landmark genome scanning and high-throughput analysis of LOH are now accelerating high-resolution genome aberration localization in human tumors. These techniques are complemented by procedures for detection of differentially expressed genes such as differential display, nucleic acid subtraction, serial analysis of gene expression and expression microarray analysis. These efforts are enabled by work from the human genome program in physical map development, cDNA library production/sequencing and in genome sequencing. This review covers several commonly used large-scale genome and gene expression analysis techniques, outlines genomic approaches to gene discovery and summarizes information that has come from large-scale analyses of human solid tumors.

Gene Dosage↗

PubMed2006

Gene expression differences in normal esophageal mucosa associated with regression and progression of mild and moderate squamous dysplasia in a high-risk Chinese population.

A randomized, double-blinded, placebo-controlled 2 x 2 factorial chemoprevention trial was conducted in Linxian, China to assess the effects of selenomethionine and celecoxib on the natural history of esophageal squamous dysplasia. Results from this study indicated that asymptomatic adults with mild dysplasia were more likely to show an improvement when treated with selenomethionine compared with placebo (P = 0.02). Prompted by this finding, we examined the molecular profiles associated with regression and progression of dysplastic lesions in normal mucosa from 29 individuals, a subset of the Linxian cohort, using the Affymetrix U133A chip. Twenty differentially expressed genes were associated with regression and 129 were associated with progression when we compared the change in gene expression over time. Genes associated with immune response (n = 15), cell cycle (n = 15), metabolism (n = 15), calcium transport or calcium ion activity (n = 10), regulation of transcription (n = 9), signal transduction (n = 7), cytoskeleton and microtubules (n = 5), nucleotide processing and biosynthesis (n = 4), G-coupled signaling (n = 4), and apoptosis (n = 3) were present in the list of 149 genes. Using the Expression Analysis Systematic Explorer pathway analysis program, only the immune response pathway was significantly overrepresented among these 149 genes. Individuals whose lesions regressed seemed to have higher expression of genes associated with immune stimulation, such as antigen presentation, survival of T cells, and T-cell activation (HLA-DRA, HLA-DPA1, HLA-DBQ1, CD58, and FCER1A). In contrast, individuals whose lesions progressed had higher expression of genes involved in immune suppression and inflammation (CNR2, NFATC4, NFRKB, MBP, INHBB, CMKLR1, CRP, ORMS, SERPINA7, and SERPINA1). These data suggest that local and systemic immune responses may influence the natural history of esophageal squamous dysplasia.

Carcinoma, Squamous Cell↗

PubMed2005

Profiling of differentially expressed genes in wound margin biopsies of horses using suppression subtractive hybridization.

Disturbed gene expression may disrupt the normal process of repair and lead to pathological situations resulting in excessive scarring. To prevent and treat impaired healing, it is necessary to first define baseline gene expression during normal repair. The objective of this study was to compare gene expression in normal intact skin (IS) and wound margin (WM) biopsies using suppression subtractive hybridization (SSH) to identify genes differentially expressed during wound repair in horses. Tissue samples included both normal IS and biopsies from 7-day-old wounds. IS cDNAs were subtracted from WM cDNAs to establish a subtracted (WM-IS) cDNA library; 226 nonredundant cDNAs were identified. Detection of genes previously shown to be expressed 7 days after trauma, including the pro-alpha(2)-chain of type 1 pro-collagen (COL1A2), annexin A(2), the pro-alpha(3)-chain of type 6 pro-collagen, beta-actin, fibroblast growth factor 7, laminin receptor 1, matrix metalloproteinase 1 (MMP1), secreted protein acidic cystein rich, and tissue inhibitor of metalloproteinase 2, supported the validity of the experimental design. A RT-PCR assay confirmed an increase or induction of the cDNAs of specific genes (COL1A2, MMP1, dermatan sulfate proteoglycan 2, cluster differentiation 68, cluster differentiation 163, and disintegrin and metalloproteinase domain 9) within wound biopsies. Among these, COL1A2 and MMP1 had previously been documented in horses; 68.8% of the cDNAs had not previously been attributed a role during wound repair, of which spermidine/spermine-N-acetyltransferase, serin proteinase inhibitor B10, and sorting nexin 9 were highly expressed and whose known functions in other processes made them potential candidates in regulating the proliferative response to wounding. In conclusion, we identified novel genes that are differentially expressed in equine wound biopsies and that may modulate repair. Future experiments must correlate changes in mRNA levels for precise molecules with spatiotemporal protein expression within tissues.

Animals↗

PubMed2005

AnovArray: a set of SAS macros for the analysis of variance of gene expression data.

BACKGROUND: Analysis of variance is a powerful approach to identify differentially expressed genes in a complex experimental design for microarray and macroarray data. The advantage of the anova model is the possibility to evaluate multiple sources of variation in an experiment. RESULTS: AnovArray is a package implementing ANOVA for gene expression data using SAS statistical software. The originality of the package is 1) to quantify the different sources of variation on all genes together, 2) to provide a quality control of the model, 3) to propose two models for a gene's variance estimation and to perform a correction for multiple comparisons. CONCLUSION: AnovArray is freely available at http://www-mig.jouy.inra.fr/stat/AnovArray and requires only SAS statistical software.

Algorithms↗

PubMed2004

Regulation of ultraviolet light-induced gene expression by gene size.

UV light induces the expression of a wide variety of genes. At present, it is unclear how cells sense the extent of DNA damage and alter the expression of UV-induced genes appropriately. UV light induces DNA damage that blocks transcription, and the probability that a gene sustains transcription-blocking DNA damage is proportional to locus size and dose of UV light. Using colon carcinoma cells that express a temperature-sensitive variant of p53 and undergo p53-dependent apoptosis after UV irradiation, we found that the number of p53-induced genes identified by oligonucleotide microarray analysis decreased in a UV dose-dependent manner. This was associated with a statistically significant shift in the spectrum of p53-induced genes toward compact genes with fewer and smaller introns. Genes encoding proapoptotic proteins involved in the initiation of the mitochondrial apoptotic cascade were prominent among the compact p53 target genes, whereas genes encoding negative regulators of p53 and the mitochondrial apoptotic pathway were significantly larger. We propose that the shift in spectrum of UV-responsive gene expression caused by passive effects of UV lesions on transcription acts as a molecular dosimeter, ensuring the elimination of cells sustaining irreparable transcription-blocking DNA damage.

Base Sequence↗

PubMed1998

Codon usage in highly expressed genes of Haemophillus influenzae and Mycobacterium tuberculosis: translational selection versus mutational bias.

Biases in the codon usage and base compositions at three codon sites in different genes of A+T-rich Gram-negative bacterium Haemophillus influenzae and G+C-rich Gram-positive bacterium Mycobacterium tuberculosis have been examined to address the following questions: (1) whether the synonymous codon usage in organisms having highly skewed base compositions is totally dictated by the mutational bias as reported previously (Sharp, P.M., Devine, K.M., 1989. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do 'prefer' optimal codons. Nucleic Acids Res. 17, 5029-5039), or is also controlled by translational selection; (2) whether preference of G in the first codon positions by highly expressed genes, as reported in Escherichia coli (Gutierrez, G., Marquez, L., Marin, A., 1996. Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency. Nucleic Acids Res. 24, 2525-2527), is true in other bacteria; and (3) whether the usage of bases in three codon positions is species-specific. Result presented here show that even in organisms with high mutational bias, translational selection plays an important role in dictating the synonymous codon usage, though the set of optimal codons is chosen in accordance with the mutational pressure. The frequencies of G-starting codons are positively correlated to the level of expression of genes, as estimated by their Codon Adaptation Index (CAI) values, in M. tuberculosis as well as in H. influenzae in spite of having an A+T-rich genome. The present study on the codon preferences of two organisms with oppositely skewed base compositions thus suggests that the preference of G-starting codons by highly expressed genes might be a general feature of bacteria, irrespective of their overall G+C contents. The ranges of variations in the frequencies of individual bases at the first and second codon positions of genes of both H. influenzae and M. tuberculosis are similar to those of E. coli, implying that though the composition of all three codon positions is governed by a selection-mutation balance, the mutational pressure has little influence in the choice of bases at the first two codon positions, even in organisms with highly biased base compositions.

Animals↗

PubMed1998

Erythroid Krüppel-like factor is essential for beta-globin gene expression even in absence of gene competition, but is not sufficient to induce the switch from gamma-globin to beta-globin gene expression.

Different genes in the beta-like globin locus are expressed at specific times during development. This is controlled, in part, by competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a specific and substantial decrease in expression of the fetal/adult-stage-specific beta-globin gene. In transgenic mice carrying the complete human beta-globin locus, EKLF ablation not only impairs human beta-globin-gene expression but also results in increased expression of the human gamma-globin genes during the fetal/adult stages. Hence, it may appear that EKLF is a determining factor for the developmental switch from gamma-globin to beta-globin transcription. However, we show here that the function of EKLF for beta-globin-gene expression is necessary even in absence of gene competition. Moreover, EKLF is not developmental specific and is present and functional before the switch from gamma-globin to beta-globin-gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of gamma-globin transcription that occurs during late fetal development is likely to be the initiating event that induces the switch.

Animals↗

PubMed1985

Screening of lambda library for differentially expressed genes using in vitro transcripts.

We present an improved approach to the screening of eucaryotic libraries for differential gene expression. Previous techniques have generated probe via the harvesting of cellular poly(A)+ RNA and synthesizing labeled cDNA probe using reverse transcriptase. In our approach we prepare labeled RNA probe via in vitro transcription. Unlike cDNA preparation, in vitro transcription (i) directly reflects the ongoing rate of gene expression, and (ii) allows one to assess expression of genes whose transcripts are not polyadenylated. To make this approach practical for the screening of a large library, we modified and optimized existing in vitro transcription techniques, enhancing manyfold the [alpha-32P]UTP incorporation into mRNA, while almost completely suppressing rRNA incorporation. In addition, we developed a simple procedure for making precise replicate dot blots of very large quantities of lambda-phage library DNA. By combining our techniques of in vitro transcription and replicate blotting, we are able to approach detection of a twofold difference in gene expression over a greater than 1000-fold range in overall expression. Our single-clone amplification and dot-blotting technique resulted in nearly the same number of lambda-phage DNA copies per dot for all members of the library. This feature allows us to assign genes to different expression classes, as well as to detect any alterations in expression. We demonstrate our approach by screening the drosophila genomic DNA library with in vitro transcripts from drosophila tissue culture cells. Screening of the entire drosophila genomic library at the 99% probability level is readily achieved.

Animals↗

PubMed2006

Bayesian robust inference for differential gene expression in microarrays with multiple samples.

We consider the problem of identifying differentially expressed genes under different conditions using gene expression microarrays. Because of the many steps involved in the experimental process, from hybridization to image analysis, cDNA microarray data often contain outliers. For example, an outlying data value could occur because of scratches or dust on the surface, imperfections in the glass, or imperfections in the array production. We develop a robust Bayesian hierarchical model for testing for differential expression. Errors are modeled explicitly using a t-distribution, which accounts for outliers. The model includes an exchangeable prior for the variances, which allows different variances for the genes but still shrinks extreme empirical variances. Our model can be used for testing for differentially expressed genes among multiple samples, and it can distinguish between the different possible patterns of differential expression when there are three or more samples. Parameter estimation is carried out using a novel version of Markov chain Monte Carlo that is appropriate when the model puts mass on subspaces of the full parameter space. The method is illustrated using two publicly available gene expression data sets. We compare our method to six other baseline and commonly used techniques, namely the t-test, the Bonferroni-adjusted t-test, significance analysis of microarrays (SAM), Efron's empirical Bayes, and EBarrays in both its lognormal-normal and gamma-gamma forms. In an experiment with HIV data, our method performed better than these alternatives, on the basis of between-replicate agreement and disagreement.

Bayes Theorem↗