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Results for “expression profiling”

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PubMed2000

Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays.

Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

Cells, Cultured↗

PubMed2006

Gene expression profiling separates chromophobe renal cell carcinoma from oncocytoma and identifies vesicular transport and cell junction proteins as differentially expressed genes.

PURPOSE: To compare gene expression profiles of chromophobe renal cell carcinoma (RCC) and benign oncocytoma, aiming at identifying differentially expressed genes. EXPERIMENTAL DESIGN: Nine cases each of chromophobe RCC and oncocytoma were analyzed by oligonucleotide microarray. Candidate genes that showed consistent differential expression were validated by reverse transcription-PCR using 25 fresh-frozen and 15 formalin-fixed, paraffin-embedded tumor samples. Immunohistochemical analysis was also done for two selected gene products, claudin 8 and MAL2. RESULTS: Unsupervised hierarchical clustering separated the chromophobe RCC and oncocytoma into two distinct groups. By a combination of data analysis approaches, we identified 11 candidate genes showing consistent differential expression between chromophobe RCC and oncocytoma. Five of these genes, AP1M2, MAL2, PROM2, PRSS8, and FLJ20171, were shown to effectively separate these two tumor groups by quantitative reverse transcription-PCR using fresh tissue samples, with similar trends seen on formalin-fixed tissues. Immunohistochemical analysis revealed selective expression of MAL2 and claudin 8 in distal renal tubules, with MAL2 antibody showing differential expression between chromophobe RCC and oncocytoma. Functional analyses suggest that genes encoding tight junction proteins and vesicular membrane trafficking proteins, normally expressed in distal nephrons, are retained in chromophobe RCC and lost or consistently down-regulated in oncocytoma, indicating that these two tumor types, believed to be both derived from distal tubules, are likely distinctive in their histogenesis. CONCLUSIONS: We showed that chromophobe RCC and oncocytoma are distinguishable by mRNA expression profiles and a panel of gene products potentially useful as diagnostic markers were identified.

Adaptor Protein Complex 1↗

PubMed2006

Expression profiling of peripheral-blood mononuclear cells from patients with chronic hepatitis C undergoing interferon therapy.

BACKGROUND: Interferon (IFN) is now the standard treatment for chronic hepatitis C (CH-C); however, treatment efficacy is unpredictable before IFN therapy is started. METHODS: We investigated the gene-expression profiles of peripheral-blood mononuclear cells (PBMCs) from patients with CH-C showing different responses to IFN. Gene-expression profiles of PBMCs were analyzed in 21 patients with CH-C treated with IFN alone or in combination with ribavirin as well as in 6 healthy volunteers. Serial changes in the gene-expression profiles of PBMCs from individual patients were evaluated before treatment, 2 weeks after the start of IFN therapy, and 6 months after the completion of IFN therapy. RESULTS: Interestingly, the gene-expression profiles of PBMCs from patients with CH-C and healthy volunteers differed substantially; early T cell-activation antigen CD69 was significantly up-regulated in patients with CH-C, but immune-related molecules such as chemokine (C-C motif) receptor 2 and interleukin 7 receptor were significantly down-regulated. Selected combinations of expressed genes obtained before treatment and during IFN therapy by use of a fuzzy neural network combined with the SWEEP operator method predicted the outcome of IFN therapy with peak accuracies of 91.0% and 90.2%, respectively. CONCLUSIONS: These findings suggest that the gene-expression profiles of PBMCs from patients with CH-C may be useful biomarkers for IFN therapy.

Adult↗

PubMed2002

Tumor specific gene expression profiles in human leiomyosarcoma: an evaluation of intratumor heterogeneity.

Leiomyosarcomas are malignant smooth muscle tumors characterized by a spectrum of histopathologic features and clinical behavior. Gene expression profiling of leiomyosarcomas may identify differential gene signatures that may allow for the clinical stratification of the tumors. Typically, surgical specimens from these tumors are large and manifest a variegated macroscopic appearance. Because of their large size at the time of diagnosis, sufficient tissue is available for regional and clonal heterogeneity assessment. However, if the gene expression profiles of samples taken from different locations in the tumors are drastically different, biologic classification on the basis of random sample analysis may not be adequate. Therefore, to assess intertumor and intratumor heterogeneity, the authors performed a gene expression study using leiomyosarcoma specimens from three excised sarcomas from an equal number of different patients. Comparisons between tumor and normal samples from the three patients as well as between carefully mapped peripheral and core specimens from the same tumor (excised from one of the patients), were performed. Analysis of the expression profiles demonstrated minimal intratumor variations compared with intertissue variations, indicating homogeneous tumor specific gene expression profiles. The authors also identified genes that are expressed differentially in tumor and normal tissue.

Biomarkers, Tumor↗

PubMed2005

Gene-expression profiles for five key glycosylation genes for galactose-fed CHO cells expressing recombinant IL-4/13 cytokine trap.

Recombinant protein glycosylation profiles have been shown to affect the in-vivo half-life, and therefore the efficacy and economics, for many therapeutics. While much research has been conducted correlating the effects of various stimuli on recombinant protein glycosylation characteristics, relatively little work has examined glycosylation-related gene-expression profiles. In this study, the effects of galactose feeding on the gene-expression profiles for five key glycosylation-related genes were determined for Chinese hamster ovary cells producing a recombinant IL-4/13 cytokine trap fusion. The genes investigated were sialidase, a putative alpha2,3-sialyltransferase, CMP-sialic acid transporter, beta1,4-galactosyltransferase, and UDP-galactosyltransferase. Additionally, the sialic acid content (sialylation) of the recombinant protein was examined. The peak sialic acid content of the IL-4/13 cytokine trap fusion protein was observed to be similar for the control and galactose-fed cultures. The gene-expression profiles for four of the glycosylation genes were observed to be sensitive to the glucose concentration and not significantly different for the control and galactose-fed cultures prior to glucose depletion. However, the sialidase gene-expression profiles were different for the control and galactose-fed cultures. The sialidase gene-expression profile increased significantly for the galactose-fed cultures prior to glucose depletion, whereas for the control cultures, the sialidase gene-expression profiles did not increase until the late stationary phase. The intracellular sialidase enzyme activity decreased exponentially with time for the control cultures; however, for the galactose-fed cultures, the intracellular sialidase enzyme activity decreased initially and then remained relatively high compared to the control cultures. These results indicate that the galactose feeding may increase the potential for desialylation, which offsets any improvements in the sialylation rate due to increased substrate levels. Thus, galactose feeding is an unnecessary expense for the production of the IL-4/13 cytokine trap fusion protein in a batch process.

Animals↗

PubMed2006

Cellular transcriptomics -- the next phase of endocrine expression profiling.

Transcriptome analysis, or global gene expression profiling, has become a commonly used and valuable tool in both basic and clinical endocrine research. Novel endocrine regulators have 'surfaced' and greater consideration is now given to understanding function at the level of gene networks. Recent developments have shown that the transcriptome is considerably larger and more divergently expressed than was previously thought. Endocrine cells express a great variety of coding and noncoding RNAs in a highly cell-specific manner. If further value is to be taken from this research area, then steps towards defined cellular transcriptomics must be taken. New sampling techniques that utilize novel genetic models are a key first step.

Animals↗

PubMed2003

Optimized protocol for linear RNA amplification and application to gene expression profiling of human renal biopsies.

Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as starting material. Standard array hybridization protocols require 5-15 micrograms labeled RNA. To obtain these quantities from small amounts of starting RNA material, RNA can be amplified in a linear fashion. Here we introduce an optimized protocol for rapid and easy-to-use amplification of as little as 1 ng total RNA. Our analysis shows that this method is linear and highly reproducible and that it preserves similarities as well as dissimilarities between normal and disease-related samples. We applied this technique to the RNA expression profiling of human renal allograft biopsies with normal histology and compared them to the profiles of renal biopsies with histological evidence of chronic transplant nephropathy or chronic rejection. Among others, complement component C1r was found to be significantly up-regulated in chronic rejection and chronic transplant nephropathy biopsies compared to normal samples, while fructose-1,6-biphosphatase showed lower-than-normal expression.

Gene Expression Profiling↗

PubMed2003

Molecular features of non-B, non-C hepatocellular carcinoma: a PCR-array gene expression profiling study.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) usually develops following chronic liver inflammation caused by hepatitis C or B virus. Through expression profiling in a rare type of HCC, for which the causes are unknown, we sought to find key genes responsible for each step of hepatocarcinogenesis in the absence of viral influence. METHODS: We used 68 non-B, non-C liver tissues (20 HCC, 17 non-tumor, 31 normal liver) for expression profiling with PCR-array carrying 3072 genes known to be expressed in liver tissues. To select the differentially expressed genes, we performed random permutation testing. A weighted voting classification algorithm was used to confirm the reliability of gene selection. We then compared these genes with the results of previous expression profiling studies. RESULTS: A total of 220 differentially expressed genes were selected by random permutation tests. The classification accuracies using these genes were 91.8, 92.0 and 100.0% by a leave-one-out cross-validation, an additional PCR-array dataset and a Stanford DNA microarray dataset, respectively. By comparing our results with previous reports on virus-infected HCC, four genes (ALB, A2M, ECHS1 and IGFBP3) were commonly selected in some studies. CONCLUSIONS: The 220 differentially expressed genes selected by PCR-array are potentially responsible for hepatocarcinogenesis in the absence of viral influence.

Aged↗

PubMed2006

Whole transcriptome amplification for gene expression profiling and development of molecular archives.

Expression profiling of clinically obtainable tumor specimens has been hindered by the need for microgram quantities of RNA. In vitro transcription (IVT)-based amplifications are most commonly used to amplify small quantities of RNA for microarray analysis. However, significant drawbacks exist with IVT-based amplification, and the need for alternative amplification methods remains. Herein, we validate whole transcriptome amplification (WTA), an exponential amplification technique that produces cDNA libraries and amplified target in 3 to 4 hours from nanogram quantities of total RNA using a combination of cDNA microarrays and quantitative polymerase chain reaction (PCR). We demonstrate that WTA material can serve as a "molecular archive" because a WTA cDNA library can be faithfully amplified through multiple rounds of PCR amplification, allowing it to serve as a bankable and distributable resource. To demonstrate applicability, WTA was combined with laser capture microdissection to profile frozen prostate tissues. Unlike most IVT-based and exponential amplification techniques, WTA does not depend on the presence of a poly-A tail. Thus, we demonstrate that WTA is compatible with artificially degraded RNA and RNA isolated from formalin-fixed paraffin-embedded tissues. Taken together, WTA represents a versatile approach to profile and archive cDNA from minute tumor samples and is compatible with partially degraded RNA.

Biological Specimen Banks↗

PubMed1999

[Gene expression profiles in squamous esophageal cancer tissues and adjacent almost normal tissues].

OBJECTIVE: To describe an esophageal cancer-specific expression profile and to identify genes that showed altered expression in squamous esophageal cancer tissues and their adjacent almost normal tissues. METHODS: The cDNA probes were synthesized from polyA(+)RNA of cancer and adjacent almost normal tissues and were differentially hybridized with two identical Atlas human cDNA expression arrays membranes containing 588 known genes. RESULTS: Autoradiographic analysis showed that of the 588 genes analyzed, 61 were found up-regulated in cancer, including cdc25B, Notch1, MMP, MET etc, and 22 down regulated in cancer, including cytokeratin4, BAD, IL-1 RECEPTOR ANTAGONIST, IL-6, etc. Expression levels of genes that associated with the regulation of cell proliferation, apoptosis, differentiation and metastasis altered most. CONCLUSION: The results for the first time provide an esophageal cancer-specific expression profile, showing that complex alterations of gene expression underlie the development of malignant phenotype of esophageal cancer cells. In addition, this line of research can lead to the identification of EC-specific genes which may be helpful for the development of diagnostic and prognostic biomarkers or therapeutic targets. The differential hybridization technique of Atlas Human cDNA expression array can be a useful method for describing the expression profiles of a tissue of cell interested.

DNA, Complementary↗

PubMed2002

Gene-expression profile comparisons distinguish seven organs of maize.

BACKGROUND: A maize array was fabricated with 5,376 unique expressed sequence tag (EST) clones sequenced from 4-day-old roots, immature ears and adult organ cDNA libraries. To elucidate organ relationships, relative mRNA levels were quantified by hybridization with embryos, three maize vegetative organs (leaf blades, leaf sheaths and roots) from multiple developmental stages, husk leaves and two types of floral organs (immature ears and silks). RESULTS: Clustering analyses of the hybridization data suggest that maize utilizes both the PEPCK and NADP-ME C(4) photosynthetic routes as genes in these pathways are co-regulated. Husk RNA has a gene-expression profile more similar to floral organs than to vegetative leaves. Only 7% of the genes were highly organ specific, showing over a fourfold difference in at least one of 12 comparisons and 37% showed a two- to fourfold difference. The majority of genes were expressed in diverse organs with little difference in transcript levels. Cross-hybridization among closely related genes within multigene families could obscure tissue specificity. As a first step in elucidating individual gene-expression patterns, we show that 45-nucleotide oligo probes produce signal intensities and signal ratios comparable to PCR probes on the same matrix. CONCLUSIONS: Gene-expression profile studies with cDNA microarrays provide a new molecular tool for defining plant organs and their relationships and for discovering new biological processes in silico. cDNA microarrays are insufficient for differentiating recently duplicated genes. Gene-specific oligo probes printed along with cDNA probes can query individual gene-expression profiles and gene families simultaneously.

Cluster Analysis↗

PubMed2003

Expression profiles of pancreatic cancer cell lines infected with antisense K-ras-expressing adenoviral vector.

The point mutations of the K-ras gene occur in as high as 70-90% of the cases with adenocarcinoma of the pancreas and apparently represent one of the key and early events in the carcinogenesis. However, the specific influence of the K-ras activation on global gene expression profiles in pancreatic cancer cells has not been elucidated. In this study, to promote elucidation of the K-ras-triggered molecular cascade(s) in pancreatic cancer, four pancreatic cancer cell lines with K-ras point mutations were infected with an adenovirus vector expressing an antisense K-ras RNA (AxCA-AS), and the change of gene expression was analyzed by oligonucleotide-based microarrays containing 12,626 genes. Among the genes showing more than 2-fold differences in the expression levels between the control- and antisense-K-ras-transduced cells, 7 genes were commonly up-regulated and 4 genes were commonly down-regulated in three or all of the four pancreatic cancer cell lines transduced with AxCA-AS. The altered gene expression levels observed by microarrays were confirmed by real-time RT-PCR methods. Then, the expression of the 4 down-regulated genes was examined in the untransduced surgical specimens of pancreatic cancer. The G-protein coupled receptor RE2 and phenylethanolamine N-methyltransferase had negligible expression levels in all pancreatic cancers, whereas the syntaxin 1A and p120 catenin isoform were significantly up-regulated in pancreatic cancers containing K-ras mutations compared with a pancreatic cancer with wild type K-ras gene. The transcriptional regulation of those genes may be a part of the molecular cascades triggered by K-ras activation leading to the development and/or progression of pancreatic cancer.

Adenoviridae↗

PubMed2003

Two azuki bean XTH genes, VaXTH1 and VaXTH2, with similar tissue-specific expression profiles, are differently regulated by auxin.

To gain insight into the functional diversity of the XTH (xyloglucan endotransglucosylase/hydrolase) gene family, we analyzed the expression profiles of two azuki bean genes, VaXTH1 and VaXTH2, which share a striking resemblance in their amino acid sequences. The two XTH genes exhibit essentially similar tissue-specific expression profiles, in that both mRNAs are found predominantly in the phloem fibers of growing internodes. However, their expression profiles are not identical. Whereas VaXTH1 is expressed in xylem cells in the basal part of the internode, little or no expression of VaXTH2 is found in the xylem. Furthermore, they exhibit spatially divergent RNA distribution profiles along the internode, VaXTH1 being expressed nearer to the top of the internode than VaXTH2. This indicates their temporally divergent expression profiles during development of the phloem fiber. Indole-3-acetic acid (IAA) up-regulates both of the mRNA levels. However, this effect of IAA on the VaXTH1 gene is nullified in 0.25 M mannitol, which prevents cell expansion without affecting auxin action per se. In contrast, the IAA-induced up-regulation of the VaXTH2 gene is not affected by mannitol. Furthermore, fusicoccin, which promotes acidification and growth, up-regulates VaXTH1 expression, but not VaXTH2 expression. Thus, the two XTH genes are committed to different steps of the cell wall dynamics in the same cell type at different stages of phloem fiber development, and are regulated by IAA in different ways.

Fabaceae↗

PubMed2006

Surface-antigen expression profiling of B cell chronic lymphocytic leukemia: from the signature of specific disease subsets to the identification of markers with prognostic relevance.

Studies of gene expression profiling have been successfully used for the identification of molecules to be employed as potential prognosticators. In analogy with gene expression profiling, we have recently proposed a novel method to identify the immunophenotypic signature of B-cell chronic lymphocytic leukemia subsets with different prognosis, named surface-antigen expression profiling. According to this approach, surface marker expression data can be analysed by data mining tools identical to those employed in gene expression profiling studies, including unsupervised and supervised algorithms, with the aim of identifying the immunophenotypic signature of B-cell chronic lymphocytic leukemia subsets with different prognosis. Here we provide an overview of the overall strategy employed for the development of such an "outcome class-predictor" based on surface-antigen expression signatures. In addition, we will also discuss how to transfer the obtained information into the routine clinical practice by providing a flow-chart indicating how to select the most relevant antigens and build-up a prognostic scoring system by weighing each antigen according to its predictive power. Although referred to B-cell chronic lymphocytic leukemia, the methodology discussed here can be also useful in the study of diseases other than B-cell chronic lymphocytic leukemia, when the purpose is to identify novel prognostic determinants.

Journal Article↗

PubMed2006

Identification of conserved modes of expression profiles during hippocampal development and neuronal differentiation in vitro.

Gene expression profiles can be regarded as sums of simpler modes, analogous to the modes of a vibrating violin string. Decomposition of temporal gene expression profiles into modes by singular value decomposition (SVD) was reported before, but the question as to what degree the SVD modes can be interpreted in terms of biology remains open. We report and compare the results of SVD of published datasets from hippocampal development, neuronal differentiation in vitro, and a control time-series hippocampal dataset. We demonstrate that the first SVD mode reflects the magnitude of expression, interpretable on the Affymetrix platform. In the datasets from gene profiling of hippocampal development and neuronal differentiation, the second mode reflects a monotonous change in expression, either up- or down-regulation, in the time course of experiment. We demonstrate that the top two SVD modes are conserved between datasets and therefore, likely reflect properties of the underlying system (gene expression in hippocampus) rather than of a particular experiment or dataset. Our results also indicate that the magnitude of expression, and the direction of change in expression during hippocampal development, are uncorrelated, suggesting that they are regulated by largely independent mechanisms.

Animals↗

PubMed2004

Expression profiling of the influence of RAS mutants on the TGFB1-induced phenotype of the pancreatic cancer cell line PANC-1.

Expression profiling analyses were used to elucidate the functional relevance of RAS proteins in mediating the effect of TGFB1 on the transcriptional phenotype of the pancreatic cancer cell line PANC-1. Despite the presence of one mutated KRAS2 allele in parental PANC-1 pancreatic cancer cells, RAS-dependent signal transduction remained susceptible to stimulation by EGF and TGFB1. To analyze the impact of RAS proteins on the TGFB1-induced transcriptional phenotype, we used PANC-1 cells stably transfected with a dominant negative HRAS(S17N) mutant or with a constitutively active KRAS2(G12V) mutant. TGFB1 treatment of mock-transfected PANC-1 cells led to an expression profile suggestive of epithelial-mesenchymal transdifferentiation (EMT). Profiling of the HRAS(S17N)-expressing clone demonstrated that induction of endogenous RAS activity by TGFB1 is required for the development of the TGFB1-induced transcriptional phenotype of PANC-1 cells. The expression of the KRAS2(G12V) mutant by itself repressed transcription of markers of epithelial differentiation and induced transcription of several extracellular matrix-associated genes. This effect was not enhanced further by TGFB1 treatment. In contrast, transcript levels of genes associated with proliferation and cell cycle progression did not appear to be the primary targets of the synergism between the RAS- and TGFB1-dependent cascades. The introduction of the dominant negative and the constitutively active RAS mutants induced partly overlapping and partly inverse effects on the TGFB1-induced expression profile of PANC-1 cells. Additional mechanisms such as the induction of autocrine loops and the use of different RAS isoforms or alternate, ERK-independent signaling pathways may be involved in the interaction between the RAS- and the TGFB1-dependent signaling cascades.

Amino Acid Substitution↗

PubMed2004

Gene expression profiles post Roux-en-Y gastric bypass.

BACKGROUND: The hypothalamus is involved in regulation of food intake (FI) and fat deposition. Molecular mechanisms of weight loss after Roux-en-Y gastric bypass (RYGB) were studied by correlating changes in gene expression profiles in hypothalamic arcuate nucleus (ARC) and subcutaneous abdominal fat (SAF). METHOD: Diet-induced obese rats were divided into RYGB, sham-operated (SO-Obese), and sham-operated pair-fed (PF) groups. A non-obese group on a regular chow diet served as control. Body weight (BW) and FI were measured. Rats were killed 10 days after the operation. Plasma was analyzed for biochemical indices, ARC and SAF were analyzed for gene expression profiles. Body SAF was also weighed. Data were analyzed by ANOVA and factor analysis. RESULTS: BW and FI decreased in RYGB versus SO-Obese, as reflected by decreased SAF (53%). Genes similarly expressed in ARC and SAF after RYGB were limited to several genes that predominantly related to metabolic pathways of carbohydrate, fat, neuropeptide, and cytokines. These expression profiles were similar to those seen in chow control and to those seen in a comparison of PF and SO-Obese. CONCLUSIONS: RYGB-induced weight loss is associated with changes in gene profile expressions that could influence metabolic changes, contributing to weight loss.

Anastomosis, Roux-en-Y↗

PubMed2006

Cell-type specific gene expression profiles of leukocytes in human peripheral blood.

BACKGROUND: Blood is a complex tissue comprising numerous cell types with distinct functions and corresponding gene expression profiles. We attempted to define the cell type specific gene expression patterns for the major constituent cells of blood, including B-cells, CD4+ T-cells, CD8+ T-cells, lymphocytes and granulocytes. We did this by comparing the global gene expression profiles of purified B-cells, CD4+ T-cells, CD8+ T-cells, granulocytes, and lymphocytes using cDNA microarrays. RESULTS: Unsupervised clustering analysis showed that similar cell populations from different donors share common gene expression profiles. Supervised analyses identified gene expression signatures for B-cells (427 genes), T-cells (222 genes), CD8+ T-cells (23 genes), granulocytes (411 genes), and lymphocytes (67 genes). No statistically significant gene expression signature was identified for CD4+ cells. Genes encoding cell surface proteins were disproportionately represented among the genes that distinguished among the lymphocyte subpopulations. Lymphocytes were distinguishable from granulocytes based on their higher levels of expression of genes encoding ribosomal proteins, while granulocytes exhibited characteristic expression of various cell surface and inflammatory proteins. CONCLUSION: The genes comprising the cell-type specific signatures encompassed many of the genes already known to be involved in cell-type specific processes, and provided clues that may prove useful in discovering the functions of many still unannotated genes. The most prominent feature of the cell type signature genes was the enrichment of genes encoding cell surface proteins, perhaps reflecting the importance of specialized systems for sensing the environment to the physiology of resting leukocytes.

Adult↗