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Results for “expression analysis”

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PubMed2005

Comparative gene expression analysis by differential clustering approach: application to the Candida albicans transcription program.

Differences in gene expression underlie many of the phenotypic variations between related organisms, yet approaches to characterize such differences on a genome-wide scale are not well developed. Here, we introduce the "differential clustering algorithm" for revealing conserved and diverged co-expression patterns. Our approach is applied at different levels of organization, ranging from pair-wise correlations within specific groups of functionally linked genes, to higher-order correlations between such groups. Using the differential clustering algorithm, we systematically compared the transcription program of the fungal pathogen Candida albicans with that of the model organism Saccharomyces cerevisiae. Many of the identified differences are related to the differential requirement for mitochondrial function in the two yeasts. Distinct regulation patterns of cell cycle genes and of amino acid metabolic genes were also revealed and, in some cases, could be linked to the differential appearance of cis-regulatory elements in the gene promoter regions. Our study provides a comprehensive framework for comparative gene expression analysis and a rich source of hypotheses for uncharacterized open reading frames and putative cis-regulatory elements in C. albicans.

Candida albicans↗

PubMed2006

Whole genome expression analysis for biologic rational pathway modeling: application in cancer prognosis and therapy prediction.

Using semi-quantitative microarray technology, almost every one of the approximately 30 000 human genes can be analyzed simultaneously with a low rate of false-positives, a high specificity, and a high quantification accuracy. This is supported by data from comparative studies of microarrays and reverse-transcription PCR for established cancer genes including those for epidermal growth factor receptor (EGFR), human epidermal growth factor receptor-2 (HER2/ERBB2), estrogen receptor (ESR1), progesterone receptor (PGR), urokinase-type plasminogen activator (PLAU), and plasminogen activator inhibitor-1 (SERPINE1). As such, semi-quantitative expression data provide an almost completely comprehensive background of biological knowledge that can be applied to cancer diagnostics. In clinical terms, expression profiling may be able to provide significant information regarding (i) the identification of high-risk patients requiring aggressive chemotherapy; (ii) the pathway control of therapy predictive parameters (e.g. ESR1 and HER2); (iii) the discovery of targets for biologically rational therapeutics (e.g. capecitabine and trastuzumab); (iv) additional support for decisions about switching therapy; (v) target discovery; and (vi) the prediction of the course of new therapies in clinical trials. In conclusion, whole genome expression analysis might be able to determine important genes related to cancer progression and adjuvant chemotherapy resistance, especially in the context of new approaches involving primary systemic chemotherapy. In this review, we will survey the current progress in whole genome expression analyses for cancer prognosis and prediction. Special emphasis is given to the approach of combining biostatistical analysis of expression data with knowledge of biochemical and genetic pathways.

Cluster Analysis↗

PubMed2005

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours.

We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3sigma, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3sigma, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours.

14-3-3 Proteins↗

PubMed2004

Rhabdomyosarcoma: value of myogenin expression analysis and molecular testing in diagnosing the alveolar subtype: an analysis of 109 paraffin-embedded specimens.

BACKGROUND: Identification of the alveolar subtype of rhabdomyosarcoma (ARMS) is important, because the poor prognosis associated with this subtype necessitates a modified therapeutic regimen. At present, ARMS diagnoses are made on the basis of histologic findings and the extent of myogenin immunopositivity. Nonetheless, the absence of an alveolar pattern in the solid variant, the low degree of differentiation in certain embryonal rhabdomyosarcomas (ERMS), and the increasing use of microbiopsy samples make the diagnosis of ARMS somewhat difficult. Two specific translocations have been found in ARMS, and fusion transcripts can be detected by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of paraffin-embedded tissue (PET). METHODS: To assess the value of myogenin staining and molecular testing in the diagnosis of rhabdomyosarcoma, the authors examined 109 rhabdomyosarcoma samples (45 ARMS samples and 64 ERMS samples). Real-time RT-PCR analysis of PET was performed in all 109 rhabdomyosarcomas, and RT-PCR analysis of frozen material was performed in 24 cases. RESULTS: PAX fusion transcripts were present in 44 cases (39 ARMS and 5 ERMS) and absent in 52 cases (2 ARMS and 50 ERMS). In 13 cases (4 ARMS and 9 ERMS), the results were not interpretable. Results were concordant between paired frozen and fixed tumor samples. All 35 interpretable ERMS samples that contained < 50% myogenin-positive cells failed to yield detectable PAX fusion transcripts. Of the 61 interpretable tumor samples (41 ARMS and 20 ERMS) that contained > 50% myogenin-positive cells, 44 (39 ARMS and 5 ERMS) yielded detectable PAX fusion transcripts. CONCLUSIONS: The current study demonstrates that molecular detection of PAX fusion transcripts via real-time RT-PCR analysis of PET is a sensitive and specific method for the diagnosis of ARMS and that immunohistochemical analysis of myogenin expression can be used to select cases for such molecular testing. Although RT-PCR analysis appears not to possess diagnostic value in tumors with < 50% tumor cell immunopositivity, it is strongly recommended for the diagnosis of tumors containing > 50% myogenin-positive cells.

Adolescent↗

PubMed1998

In vitro expression analysis of mutations in phenylalanine hydroxylase: linking genotype to phenotype and structure to function.

Mutations in the human phenylalanine hydroxylase gene (PAH) altering the expressed cDNA nucleotide sequence (GenBank U49897) can impair activity of the corresponding enzyme product (hepatic phenylalanine hydroxylase, PAH) and cause hyperphenylalaninemia (HPA), a metabolic phenotype for which the major disease form is phenylketonuria (PKU; OMIM 261600). In vitro expression analysis of inherited human mutations in eukaryotic, prokaryotic, and cell-free systems is informative about the mechanisms of mutation effects on enzymatic activity and their predicted effect on the metabolic phenotype. Corresponding analysis of site-directed mutations in rat Pah cDNA has assigned critical functional roles to individual amino acid residues within the best understood species of phenylalanine hydroxylase. Data on in vitro expression of 35 inherited human mutations and 22 created rat mutations are reviewed here. The core data are accessible at the PAH Mutation Analysis Consortium Web site (http://www.mcgill.ca/pahdb).

Animals↗

PubMed2004

Comprehensive expression analysis of a rat depression model.

Herein we report on a large-scale analysis of gene expression in the 'learned helplessness' (LH) rat model of human depression, using DNA microarrays. We compared gene expression in the frontal cortex (FC) and hippocampus (HPC) of untreated controls, and LH rats treated with saline (LH-S), imipramine or fluoxetine. A total of 34 and 48 transcripts were differentially expressed in the FC and HPC, respectively, between control and LH-S groups. Unexpectedly, only genes for NADH dehydrogenase and zinc transporter were altered in both the FC and HPC, suggesting limited overlap in the molecular processes from specific areas of the brain. Principal component analysis revealed that sets of upregulated metabolic enzyme genes in the FC and downregulated genes for signal transduction in the HPC can distinguish clearly between depressed and control animals, as well as explain the responsiveness to antidepressants. This comprehensive data could help to unravel the complex genetic predispositions involved in human depression.

Animals↗

PubMed1999

Molecular cloning and expression analysis of a mouse phospholipase C-delta1.

We describe here the molecular cloning and expression analysis of mouse PLC-delta1 (mPLC-delta1), a key enzyme in cell signal transduction. A mouse brain cDNA library was screened in order to isolate the mPLC-delta1 cDNA. The mPLC-delta1 cDNA was 2660 bp in length. The predicted open reading frame encodes a protein of 756 amino acids with an estimated molecular mass of 85 kDa. The deduced amino acid sequence exhibits 96.9% and 92.7% identity with the sequence of rat and human PLC-delta1, respectively. The mPLC-delta1 mRNA was highly expressed in brain, heart, lung, and testis. We found that transcripts of mPLC-delta1 are present in almost all regions of mouse brain examined, implying that the enzyme may play a role in some fundamental cellular process in brain. In male reproductive tract, mPLC-delta1 mRNA was widely expressed in the epididymis as well as in the testis. In situ hybridization studies indicate that distribution of mPLC-delta1 mRNA in mouse testis is discrete and unique. The expression of mPLC-delta1 mRNA was defined in the periphery of each seminiferous tubule, especially in spermatogonia, which might imply that mPLC-delta1 plays a role in proliferation of spermatogonia. To the best our knowledge, this is the first report to demonstrate the high expression of mPLC-delta1 mRNA in spermatogonia of testis. Taken together, these results suggest that mPLC-delta1 may carry out fundamental roles in almost all of mouse tissues, especially in brain and specific roles in testis.

Amino Acid Sequence↗

PubMed2005

Expression analysis onto microarrays of randomly selected cDNA clones highlights HOXB13 as a marker of human prostate cancer.

In a strategy aimed at identifying novel markers of human prostate cancer, we performed expression analysis using microarrays of clones randomly selected from a cDNA library prepared from the LNCaP prostate cancer cell line. Comparisons of expression profiles in primary human prostate cancer, adjacent normal prostate tissue, and a selection of other (nonprostate) normal human tissues, led to the identification of a set of clones that were judged as the best candidate markers of normal and/or malignant prostate tissue. DNA sequencing of the selected clones revealed that they included 10 genes that had previously been established as prostate markers: NKX3.1, KLK2, KLK3 (PSA), FOLH1 (PSMA), STEAP2, PSGR, PRAC, RDH11, Prostein and FASN. Following analysis of the expression patterns of all selected and sequenced genes through interrogation of SAGE databases, a further three genes from our clone set, HOXB13, SPON2 and NCAM2, emerged as additional candidate markers of human prostate cancer. Quantitative RT-PCR demonstrated the specificity of expression of HOXB13 in prostate tissue and revealed its ubiquitous expression in a series of 37 primary prostate cancers and 20 normal prostates. These results demonstrate the utility of this expression-microarray approach in hunting for new markers of individual human cancer types.

Biomarkers, Tumor↗

PubMed2006

Gene expression analysis in blood cells in response to unmodified and 2'-modified siRNAs reveals TLR-dependent and independent effects.

Ribonucleic nucleic acid recognition by Toll-like receptors (TLRs) induces innate immune responses. However, no comprehensive analysis of gene expression in human blood cells in response to unmodified and 2'-modified immunostimulatory RNAs has been reported. Using oligonucleotide microarrays, we show that around 400 genes were significantly (P<0.001) altered in peripheral blood mononuclear cells (PBMC) in response to either single-stranded (ss) or double-stranded (ds) small interfering RNAs (siRNAs). Most of the upregulated genes encode proteins involved in innate and adaptive immune responses, including proinflammatory cytokines, interferons, chemokines and chemokine receptors. Genes encoding proteins involved in lymphocyte activation (e.g. CD80, CD40, and CD69) and in regulation of the immune responses (e.g. SOCS proteins) were upregulated. Also, genes encoding for antiviral proteins (Mx1, Mx2, TRIM proteins), and interferon regulatory factors (e.g. IRF7) were upregulated. Around 90% of the genes (140 out of 160) affected by R-848, a specific ligand for TLR7 and TLR8, were also affected by ss siRNAs or ds siRNAs, indicating that the signaling pathways activated by R-848 are also activated by immunostimulatory siRNAs. In addition to immunoactivation via TLRs, ss siRNAs and ds siRNAs induced TLR-independent gene alterations. Surprisingly, replacement of only uridine bases with either 2'-fluoro or 2'-O-methyl modified counterparts abrogated all the observed bystander effects. Collectively, these microarray data offer for the first time an insight into human PMBC response to immunostimulatory RNAs such as ss siRNAs and ds siRNAs. The data should help to define strategies to either enhance or avoid the non-specific effects of siRNAs in order to develop safe therapeutics.

Gene Expression Profiling↗

PubMed2004

Responses of primary and secondary metabolism to sugar accumulation revealed by microarray expression analysis of the Arabidopsis mutant, pho3.

The Arabidopsis mutant pho3 accumulates sucrose and other carbohydrates to high levels, providing a means of investigating the genomic response to sucrose accumulation using microarray analysis. Wild-type and mutant plants were grown in soil to the mature rosette stage for the analysis of gene expression using the Affymetrix ATH1 chip, containing more than 22,500 probe sets. Small, but significant, decreases were observed in the expression of many genes encoding enzymes and regulatory proteins involved in primary carbon assimilation, suggesting that, in mature leaves of Arabidopsis, there is limited feedback regulation on gene expression by sugars. The study revealed a striking increase in the expression of the plastid glucose 6-phosphate/phosphate translocator, characteristically expressed only in heterotrophic tissues. This indicated a change in the nature of metabolite exchange between the plastid and the cytosol in the pho3 mutant. The expression of enzymes of starch synthesis also increased significantly. Very large increases were observed in the expression of transcription factors and enzymes involved in anthocyanin biosynthesis. This finding reinforces the emerging picture of an important role for primary metabolism in regulating secondary metabolism.

Arabidopsis↗

PubMed2005

Evidence by expression analysis of candidate genes for congenital heart defects in the NF1 microdeletion interval.

It was recently reported that congenital heart disease is significantly more frequent in patients with NF1 microdeletion syndrome than in those with classical NF1. The outcome of congenital heart disease in this subset of patients is likely caused by the haploinsufficiency of gene/s in the deletion interval. Following in silico analysis of the deleted region, we found two genes known to be expressed in adult heart, the Joined to JAZF1 (SUZ12) and the Centaurin-alpha 2 (CENTA2) genes, and seven other genes with poorly defined patterns of expression and function. With the aim of defining their expression profiles in human fetal tissues (15th-21st weeks of gestation), expression analysis by RT-PCR and Northern blotting was performed. C17orf40, SUZ12 and CENTA2 were found to be mainly expressed in fetal heart, and following RT-PCR on mouse embryos and embryonic heart and brain at different stages of development, we found that the orthologous genes C17orf40, Suz12 and Centa2 are also expressed in early stages of development, before and during the formation of the four heart chambers. The presence of binding sites for Nkx2-5, a transcription factor expressed early in heart development, in all three mouse orthologous genes was predicted by bioinformatics, thus reinforcing the hypothesis that these genes might be involved in heart development and may be plausible candidates for congenital heart disease.

Animals↗

PubMed2002

Large-scale gene expression analysis in molecular target discovery.

The evolution of simple arrays consisting of a few genes to ones composed of thousands of genes and/or ESTs has allowed investigators unprecedented views of the molecular mechanisms within cells. Due to the enormous quantities of information derived from microarray analysis, new types of problems have surfaced, such as where to store all of the data. The ability to solve database or statistical problems has required the bench biologist to collaborate with database developers, software designers and statisticians to determine solutions for storage, analysis and interpretation of microarray data. The collaborative effort between these extremely diverse disciplines has led to the development of creative database query and gene expression analysis tools, producing significant reductions in the time required by researchers to filter through the datasets and discover the key processes perturbed by the diseases of interest. Both unsupervised and supervised analysis methods have been applied to gene expression data leading to the discovery of novel therapeutic targets and diagnostic markers. Furthermore, tumor classification based on their respective molecular fingerprints has led to the classification of cancer subtypes and the discovery of novel molecular taxonomies that may eventually lead to improved patient stratification and superior therapeutic strategies.

Databases, Factual↗

PubMed2005

Genome-wide expression analysis reveals 100 adrenal gland-dependent circadian genes in the mouse liver.

Recent progress in genome-wide expression analysis has identified hundreds of circadian genes not only in the suprachiasmatic nucleus (the mammalian master clock) but also in peripheral tissues, such as heart, liver and kidney of mammals. Glucocorticoid is thought to be a circadian time cue for mammalian peripheral clocks. To identify the genes of which the circadian expression is regulated by endogenous glucocorticoids, we performed DNA microarray analysis using hepatic RNA from adrenalectomized (ADX) and sham-operated mice. We identified 169 genes that fluctuated between day and night in the livers of the sham-operated mice. Among these, 100 lost circadian rhythmicity in ADX mice. These included the genes for key enzymes of liver metabolic functions, such as glucokinase, HMG-CoA reductase and glucose-6-phosphatase. The circadian expression of Lpin1, FKBP51 and S-adenosyl methionine decarboxylase was also abolished in the ADX mice. On the other hand, although the circadian expression of clock or clock-related genes, such as mPer2, DBP, E4BP4, mDec1, Usp2 and Wee1 remained almost totally intact in the liver of ADX mice, it was extremely damped in homozygous Clock mutant mice. The present findings suggested that one type of hepatic circadian genes in mice is transcriptionally regulated by core components of the circadian clock, such as CLOCK and BMAL1, and that the other depends on the adrenal gland.

Adrenal Glands↗

PubMed2004

Direct gene expression analysis.

The direct analysis of single biological molecules is getting increasingly important in basic as well as pharmaceutical research (e.g. for gene expression analysis). In particular single-molecule fluorescence detection provides exciting new opportunities to probe biochemical processes in unprecedented detail. Currently several academic and industrial research groups work on the development of single molecule detection based technologies in order to directly detect and analyze RNA and DNA molecules. As these developed methods are characterized as homogenous assays and obviate any amplification of the target or the signal, they provide clear advantages compared to methods like real-time PCR or DNA- arrays. In the following we describe a recently developed approach based on fluorescence correlation spectroscopy (FCS). This expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target molecule (either DNA or RNA) in solution. The subsequent dual color cross-correlation analysis allows the quantification of the bio-molecule of interest in absolute numbers. Target concentrations of less than 10(-12) M can be easily monitored, covering the direct analysis of the expression levels of high, medium and low abundant genes.

Animals↗

PubMedDate not supplied

House keeping genes and gene expression analysis in transplant recipients: a note of caution.

BACKGROUND: House keeping genes are often used as a means of standardising results obtained in gene expression investigations. This study was performed to investigate whether beta-actin, beta2-microglobulin (two genes frequently quoted as house keeping genes) and/or transferrin receptor would be suitable house keeping genes for use in gene expression analysis of renal transplant recipients. METHODS: Sequential expression of all three genes was measured in the peripheral blood mononuclear cells of 13 living donors and 45 renal transplant recipients, pre-operatively and then daily for up to 2 weeks. Fifteen of the recipients experienced an episode of biopsy proven acute rejection. Gene expression measurement was performed using quantitative real time 'TaqMan' PCR technology. RESULTS: Gene expression of all three genes was unchanged in the living donor cohort. However, in the transplant recipients there were significant increases in expression following transplantation in the non-rejectors, and preceding the diagnosis of acute rejection. In the latter group, levels returned to pre-transplant values after the commencement of anti-rejection therapy. CONCLUSIONS: Beta-actin, beta2-microglobulin and transferrin receptor gene expression, although not influenced by surgery, is influenced by transplantation, acute rejection and anti-rejection therapy making these genes unsuitable as house keeping genes following renal transplantation. These findings may cast doubt on the results of some studies that used these genes for the purposes of standardisation when looking at cDNA measurement. We suggest that any group wishing to use a house keeping gene ensure that its expression is independent of study parameters prior to the start of the study.

Actins↗

PubMed2006

The use of gene expression analysis and proteomic databases in the development of a screening system to determine the value of natural medicinal products.

A rapid throughput screening system involving gene expression analysis was developed in order to investigate the potential of bioactive chemicals contained in natural health products as effective drug therapy, in particular the ability of these chemicals to alleviate the inflammatory response in human airway epithelial cells. A number of databases were searched to retrieve the information needed to properly analyze the gene expression profiles obtained. The gene expression of human bronchial epithelial cells infected with rhinovirus and/or exposed to platelet activating factor was analyzed. Following analysis of the gene expression data the total number of expressed proteins that may potentially act as a marker for monitoring the modulation of airway inflammation was narrowed to 19. Further studies will involve selecting antibodies for these proteins, culturing airway epithelial cells in the presence of extracts of natural health products, extracting the proteins and identifying them by western blot analysis.

Journal Article↗

PubMed1999

Molecular cloning and expression analysis of a gene for a sucrose transporter in maize (Zea mays L.).

Here we report the cloning of a sucrose transporter cDNA from maize (Zea mays L.) and an analysis of the expression of the gene. A cDNA clone (ZmSUT1) was identified as a sucrose transporter cDNA from its sequence homology at the amino acid level to sucrose transporters that have been cloned from other higher plant species. Based on the results of genomic Southern hybridization, ZmSUT1 appears to be a single copy gene. A Northern blot analysis of seedlings and leaf blades suggests that the sucrose transporter is involved in the export of accumulated carbohydrates from source leaf blades. From the measurements of transcript levels and carbohydrate contents in mature leaf blades, we propose that the expression of the gene for the maize sucrose transporter is positively regulated by the amounts of soluble sugars such as sucrose and glucose in source leaves of maize. In addition, based on the tissue specificity of the expression of the gene in maize plants at the reproductive stage, it is possible that the sucrose transporter acts in sink tissues such as pedicles as well as in source tissues such as leaf blades.

Biological Transport↗

PubMed2003

[DNA chips for gene expression analysis and their application in diagnostics].

Due to large genome projects more and more genetic information and more and more data about genetic background of various diseases can be obtained. By means of microarrays thousands to hundreds of thousands of molecular analysis can be performed at a time, rapidly, under identical conditions, hereby microarrays provide a solution to continuously increasing throughput demands from large genomic, pharmacogenetic and proteomic programmes. Microarrays offer unprecedented opportunities for qualitative and quantitative analysis of gene expression, understanding gene function and detection gene alterations, polimorphisms. Microarray expression profiles will define stages of different tumor types and new prognostic subgroups in cancer and other diseases. For individual patients, more precise diagnosis and risk assessment based on expression profiles are already achievable, leading to more accurate determination of prognosis and more individually tailored treatment. Microarrays will play an important role in diagnostics of infectious diseases and identification of novel diagnostic and therapeutic targets. Wide-spread application of microarrays in clinical practice is not typical today, but their production on an industrial scale and the increased information provided by microarrays should assure their entry into clinical practice as very powerful, sensitive, specific and rapid tools of diagnostics of various diseases.

Communicable Diseases↗