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PubMed2004

GECKO: a complete large-scale gene expression analysis platform.

BACKGROUND: Gecko (Gene Expression: Computation and Knowledge Organization) is a complete, high-capacity centralized gene expression analysis system, developed in response to the needs of a distributed user community. RESULTS: Based on a client-server architecture, with a centralized repository of typically many tens of thousands of Affymetrix scans, Gecko includes automatic processing pipelines for uploading data from remote sites, a data base, a computational engine implementing approximately 50 different analysis tools, and a client application. Among available analysis tools are clustering methods, principal component analysis, supervised classification including feature selection and cross-validation, multi-factorial ANOVA, statistical contrast calculations, and various post-processing tools for extracting data at given error rates or significance levels. On account of its open architecture, Gecko also allows for the integration of new algorithms. The Gecko framework is very general: non-Affymetrix and non-gene expression data can be analyzed as well. A unique feature of the Gecko architecture is the concept of the Analysis Tree (actually, a directed acyclic graph), in which all successive results in ongoing analyses are saved. This approach has proven invaluable in allowing a large (approximately 100 users) and distributed community to share results, and to repeatedly return over a span of years to older and potentially very complex analyses of gene expression data. CONCLUSIONS: The Gecko system is being made publicly available as free software http://sourceforge.net/projects/geckoe. In totality or in parts, the Gecko framework should prove useful to users and system developers with a broad range of analysis needs.

Carcinoma↗
PubMed2002

Ribosomal highly basic 23-kDa protein as a reliable standard for gene expression analysis.

BACKGROUND/AIMS: Analysis of gene expression is dependent on normalization using housekeeping genes. However, many of these housekeeping genes (e.g. GAPDH, beta-actin) are upregulated in chronic pancreatitis and pancreatic cancer, and cannot be used for normalization. For this reason we tried to identify a housekeeping gene useful for expression analysis in pancreatic diseases. METHODS: RNA isolated from various tissues and states of disease was subjected to reverse transcription and subsequently amplified by PCR using primers for GAPDH and for the ribosomal highly basic 23-kDa (rb 23-kDa, RPL13A) protein. RESULTS: As anticipated, expression of GAPDH varied markedly in the different tissues, whereas the expression of rb 23-kDa was constant in all samples investigated. CONCLUSION: We recommend the use of the ribosomal highly basic 23-kDa protein as a standard for normalization at least for the pancreas and the prostate.

Gene Expression↗
PubMed2005

Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization.

Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.

Animals↗
PubMed2006

Technical note: validation of internal control genes for gene expression analysis in bovine polymorphonuclear leukocytes.

Analysis of gene expression is becoming more important in all areas of biological research to evaluate gene expression during physiological and pathological conditions (e.g., mastitis), not the least in the field of animal research. Presently, real-time gene expression analysis is considered to be the method of choice for accurate and sensitive quantification of mRNA transcripts. Because comparison of gene expression levels is frequently the aim of these experiments, there is a critical need to validate internal control genes. When studying gene expression in bovine polymorphonuclear leukocytes, special attention should be paid to this validation, because polymorphonuclear leukocytes are subjected to numerous physiological influences, depending on the stage of lactation. In this study, 8 commonly used reference genes (ACT, GAPD, H2A, TBP, HPRT1, SDHA, YWHAZ, and 18S rRNA) were evaluated in bovine polymorphonuclear leukocytes. The transcription levels of 6 reference genes were determined using real-time PCR. By geometrically averaging the expression levels of these genes, SDHA, YWHAZ, and 18S rRNA were selected as being the most stable genes for accurate normalization of real-time results of bovine polymorphonuclear leukocytes.

Animals↗
PubMed2003

Global gene expression analysis of single cells.

Gene expression profiling is increasingly being used to study complex disease processes, with a focus toward generating new hypotheses and identifying novel therapeutic approaches. This method requires not only the ability to assign expression data to the correct cell type, but also the aptitude to interpret the subsequent deluge of gene expression patterns. Single-cell gene expression analysis is currently used to generate data within the fundamental unit, the single cell, thereby freeing the analysis from assumptions or questions regarding cell population homogeneity, whether cell-type or temporal. Single-cell expression profiling also offers a highly parallel view of the workings of a gene regulatory network at one specific point in time, and will hopefully provide insights that could lead to an improved ability to interpret gene expression patterns.

Animals↗
PubMedDate not supplied

Gene expression analysis of troglitazone reveals its impact on multiple pathways in cell culture: a case for in vitro platforms combined with gene expression analysis for early (idiosyncratic) toxicity screening.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists of the thiazolidinedione family are used for the treatment of type 2 diabetes mellitus due to their ability to reduce glucose and lipid levels in patients with this disease. Three thiazolidinediones that were approved for treatment are Rezulin (troglitazone), Avandia (rosiglitazone), and Actos (pioglitazone). Troglitazone was withdrawn from the market due to idiosyncratic drug toxicity. Rosiglitazone and pioglitazone are still on the market for the treatment of type 2 diabetes. The authors present data from a gene expression screen that compares the impact these three compounds have in rats, in rat hepatocytes, and in the clone 9 rat liver cell line. The authors monitored the changes in expression in multiple genes, including those related to xenobiotic metabolism, proliferation, DNA damage, oxidative stress, apoptosis, and inflammation. Compared to the other two compounds, troglitazone had a significant impact on many of the pathways monitored in vitro although no major perturbation was detected in vivo. The changes detected predict not only general toxicity but potential mechanisms of toxicity. Based on gene expression analysis, the authors propose there is not just one but multiple ways troglitazone could be toxic, depending on a patient's environment and genetic makeup, including immune response-related toxicity.

Animals↗
PubMed2006

A framework for gene expression analysis.

MOTIVATION: Global gene expression measurements as obtained, for example, in microarray experiments can provide important clues to the underlying transcriptional control mechanisms and network structure of a biological cell. In the absence of a detailed understanding of this gene regulation, current attempts at classification of expression data rely on clustering and pattern recognition techniques employing ad-hoc similarity criteria. To improve this situation, a better understanding of the expected relationships between expression profiles of genes associated by biological function is required. RESULTS: It is shown that perturbation expansions familiar from biological systems theory make precise predictions for the types of relationships to be expected for expression profiles of biologically associated genes, even if the underlying biological factors responsible for this association are not known. Classification criteria are derived, most of which are not usually employed in clustering algorithms. The approach is illustrated by using the AtGenExpress Arabidopsis thaliana developmental expression map.

Algorithms↗
PubMed2006

Genome-controlled reverse transcriptase-polymerase chain reaction for targeted gene-expression analysis.

OBJECTIVE: Although gene-expression profiling has an important part to play in the classification of tumours and premalignant conditions, reproducibility of the present polymerase chain reaction (PCR)-based quantitative techniques needs to be improved for diagnostic purposes and to enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. We have developed reverse transcriptase-PCR-based technology for quantitative assessment of the relative content of multiple mRNA transcripts in small tissue or cell samples. MATERIAL AND METHODS: A multiplexed sequence modifying cDNA synthesis reaction is performed with this technique to create a 4-5 degrees increase in the melting temperature of subsequent short (56-64 bp) PCR amplicons. Each cDNA template is competitively co-amplified with genomic DNA, which serves as a universal internal standard. The relative amounts of cDNA and genomic DNA-derived amplicons are quantified in-tube by homogeneous melting curve analysis. RESULTS: The dynamic range of the assay was three orders of magnitude, while the detection limit was 100 cDNA molecules. A prototype assay, consisting of the analysis of eight genes, displayed good reproducibility (inter-assay CV 5-20 %) compared to the TaqMan assay (inter-assay CV 7-43 %). Gene-expression analysis could be performed in 20 of 20 (100 %) archival frozen samples, in 30 of 35 (86 %) archival FFPE samples and in 26 of 27 (96 %) endoscopic biopsies. CONCLUSIONS: We demonstrate that this new technique enables accurate analysis of mRNA expression in cultured cells and endoscopic tissue biopsies. Sensitive analysis FFPE tissue is also possible thanks to the short PCR amplicons.

Biopsy↗
PubMed2006

Gene expression analysis using human cancer xenografts to identify novel predictive marker genes for the efficacy of 5-fluorouracil-based drugs.

The development of a diagnostic method for predicting the therapeutic efficacy or toxicity of anticancer drugs is a critical issue. We carried out a gene expression analysis to identify genes whose expression profiles were correlated with the sensitivity of 30 human tumor xenografts to 5-fluorouracil (5-FU)-based drugs (tegafur + uracil [UFT], tegafur + gimeracil + oteracil [S-1], 5'-deoxy-5-fluorouridine [5'-DFUR], and N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine [capecitabine]), as well as three other drugs (cisplatin [CDDP], irinotecan hydrochloride [CPT-11], and paclitaxel) that have different modes of action. In the present study, we focused especially on the fluoropyrimidines. The efficacy of all anticancer drugs was assayed using human tumor xenografts in nude mice. The mRNA expression profile of each of these xenografts was analyzed using a Human Focus array. Correlation analysis between the gene expression profiles and the chemosensitivities of seven drugs identified 39 genes whose expression levels were correlated significantly with multidrug sensitivity, and we suggest that the angiogenic pathway plays a pivotal role in resistance to fluoropyrimidines. Furthermore, many genes showing specific correlations with each drug were also identified. Among the candidate genes associated with 5-FU resistance, the dihydropyrimidine dehydrogenase mRNA expression profiles of the tumors showed a significant negative correlation with chemosensitivity to all of the 5-FU based drugs except for S-1. Therefore, the administration of S-1 might be an effective strategy for the treatment of high dihydropyrimidine dehydrogenase-expressing tumors. The results of the present study may enhance the prediction of tumor response to anticancer drugs and contribute to the development of tailor-made chemotherapy.

Animals↗
PubMed2002

[Study of the gene expression pattern in the Kkay mouse with type 2 diabetes by genomic-scale gene expression analysis].

OBJECTIVE: To study the genes differentially expressed in the liver of Kkay diabetic and normal mice by genomic-scale gene expression analysis. METHODS: cDNA microarray chips containing 8,192 cDNAs were used to explore the gene expression pattern of Kkay mouse liver. RESULTS: One hundred and fifty-four genes were screened out, including 68 complete cDNAs and expressed sequence tags, and among them 40 genes were up-regulated and 114 genes were down-regulated respectively. CONCLUSION: Most of the gene expression analysis results were consistent with previous study, and the gene expression pattern of Kkay mouse based on cDNA microarray could be used for high-throughout screening out the genes associated with type 2 diabetes.

Animals↗
PubMed2004

Validation of internal control genes for gene expression analysis in diabetic glomerulosclerosis.

BACKGROUND: Gene expression analysis is an invaluable tool in the study of diabetic glomerulosclerosis. The necessary denominator for the quantitative expression of a specific gene is the expression level of a second gene that is presumed to remain unchanged. Thus, it is critical that the stability of this housekeeping gene in diabetic glomeruli or in cultured glomerular cells is not altered by the disease or a high glucose environment, respectively. Although gene expression quantification, achieved by Northern blot analysis or real-time reverse transcription-polymerase chain reaction (RT-PCR) has been extensively applied in diabetic renal tissue in vivo and in vitro, there are no studies validating the use of any specific endogenous control gene in these measurements. METHODS: We performed real-time RT-PCR using RNA from microdissected diabetic glomeruli and from mesangial cells cultured in high glucose concentration to investigate gene expression stability of beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GADPH), phospholipase A2, beta2-microglobulin, acidic ribosomal protein 36B4, and cyclophilin A. RESULTS: Using an analysis method which is independent of gene abundance and compares the pair-wise variation of a given housekeeping gene with all other control genes, beta-actin and phospholipase A2, were found to be the most stable genes in diabetic glomeruli and in primary mesangial cells exposed to 20 mmol/L glucose. CONCLUSION: It is proposed that the expression level of these genes is the best reference to evaluate relative changes in gene activity in diabetic/high glucose exposed glomerular tissues.

Actins↗
PubMed2004

Optimizing RNA extraction yield from whole blood for microarray gene expression analysis.

OBJECTIVES: Microarray analysis of gene expression profiles of blood leukocytes has many potential clinical and research applications. DESIGN AND METHODS: We used the PAXgene Blood RNA System to prepare RNA from the whole blood of normal volunteers using two incubation times followed by gene expression profiling using the Affymetrix HU133A GeneChip. CONCLUSIONS: Longer incubation gave a significantly higher RNA yield and samples that were satisfactory for microarray analysis, with excellent pairwise correlations between replicates.

Gene Expression Profiling↗
PubMed2002

Challenges of single-cell diagnostics: analysis of gene expression.

Analysis of single-cell gene expression promises a more precise understanding of human disease pathogenesis and important diagnostic applications. Here, we review the rationale for the study of gene expression at the single-cell level, practical methods to isolate homogeneous or single-cell samples, and current approaches to the analysis of single-cell gene expression. Finally, we highlight applications of laser microdissection-based gene expression analysis to the study of human disease and clinical diagnosis.

Animals↗
PubMed2004

Comprehensive vertical sample-based KNN/LSVM classification for gene expression analysis.

Classification analysis of microarray gene expression data has been widely used to uncover biological features and to distinguish closely related cell types that often appear in the diagnosis of cancer. However, the number of dimensions of gene expression data is often very high, e.g., in the hundreds or thousands. Accurate and efficient classification of such high-dimensional data remains a contemporary challenge. In this paper, we propose a comprehensive vertical sample-based KNN/LSVM classification approach with weights optimized by genetic algorithms for high-dimensional data. Experiments on common gene expression datasets demonstrated that our approach can achieve high accuracy and efficiency at the same time. The improvement of speed is mainly related to the vertical data representation, P-tree,Patents are pending on the P-tree technology. This work is partially supported by GSA Grant ACT#:K96130308. and its optimized logical algebra. The high accuracy is due to the combination of a KNN majority voting approach and a local support vector machine approach that makes optimal decisions at the local level. As a result, our approach could be a powerful tool for high-dimensional gene expression data analysis.

Algorithms↗
PubMed2006

Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR.

Gene expression analysis provides significant insight to understand regulatory mechanisms of biology, yet acquisition and reproduction of quality data, as well as data confirmation and verification remain challenging due to a lack of proper quality controls across different assay platforms. We present a set of six universal external RNA quality controls for microbial mRNA expression analysis that can be applied to both DNA oligo microarray and real-time qRT-PCR including using SYBR Green and TaqMan probe-based chemistry. This set of controls was applied for Saccharomyces cerevisiae and Pseudomonas fluorescens Pf-5 microarray assays and qRT-PCR for yeast gene expression analysis. Highly fitted linear relationships between detected signal intensity and mRNA input were described. Valid mRNA detection range, from 10 to 7000 pg and from 100 fg to 1000 pg were defined for microarray and qRT-PCR assay, respectively. Quantitative estimation of mRNA abundance was tested using randomly selected yeast ORF including function unknown genes using the same source of samples by the two assay platforms. Estimates of mRNA abundance by the two methods were similar and highly correlated in an overlapping detection range from 10 to 1000 pg. The universal external RNA controls provide a means to compare microbial gene expression data derived from different experiments and different platforms for verification and confirmation. Such quality controls ensure reliability and reproducibility of gene expression data, and provide unbiased normalization reference for validation, quantification, and estimate of variation of gene expression experiments. Application of these controls also improves efficiency and facilitates high throughput applications of gene expression analysis using the qRT-PCR assay.

Animals↗
PubMed2003

Purification of Mycobacterium leprae RNA for gene expression analysis from leprosy biopsy specimens.

Gene expression analysis in Mycobacterium leprae, an obligate intracellular pathogen and the etiologic agent of leprosy, has been hampered by the lack of an efficient method to purify RNA from leprosy lesions. Therefore to date, transcripts for only a few genes have been identified. We report the use of a single-tube homogenization/RNA extraction method that produces enough RNA to study the expression of 30 genes from a single skin biopsy specimen of a multibacillary leprosy patient and demonstrate that RNA can be purified after fixation of biopsies in 70% ethanol for up to a year. This represents a major advancement in the ability to study M. leprae gene expression directly from biopsy material and should help to define genes that are associated with intracellular survival of this human pathogen.

Biopsy↗
PubMed2003

Selection of normalizer genes in conducting relative gene expression analysis of embryos.

BACKGROUND: In relative gene expression analysis, a reference gene for sample normalization is required for determining target expression changes among experimental treatment groups. Since some developmental toxicants secondarily cause general growth retardation and/or other general biological changes, commonly used housekeeping genes may not serve as accurate normalizers. METHODS: We conducted real-time polymerase chain reaction (PCR) with normalization to calculate relative target transcriptional change, using housekeeping and structure-specific expression genes as normalizers. Relative levels of Hoxb1 expression were measured in cultured rodent embryos at 24 hr post retinoic acid (RA) administration. Transcriptional response was also evaluated using two novel compounds that produced posterior axial and growth defects in rat whole-embryo culture. Embryos treated with these compounds were evaluated for general biological processes, and their respective biological states were considered in the context of the relative gene expression change calculated with the housekeeping normalizers. RESULTS: Normalized RA-induced Hoxb1 expression demonstrated that only some reference genes accurately quantitated the expected 1.5- to 2-fold increase in Hoxb1 expression. Evaluation of the test compounds demonstrated that only normalization with the spatially-restricted hindbrain gene, Krox-20, calculated significant expression decreases of T-gene, a gene known to be functionally relevant in posterior axial development. Reduction in T-gene expression was confirmed qualitatively by whole-mount in situ hybridization. CONCLUSIONS: Prudent reference gene selection is important in evaluating relative gene expression in embryos. An experimental control design is proposed to facilitate the identification of normalizing genes that will accurately calculate relative gene expression change in treated embryos.

Animals↗
PubMed2006

Targeted gene-expression analysis by genome-controlled reverse transcription-PCR.

BACKGROUND: For gene-expression analysis, which is anticipated to play an important role in classification of tumors and premalignant conditions, PCR-based quantitative assays must have increased diagnostic quantitative accuracy and reproducibility and enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. METHODS: We developed a reverse transcription-PCR-based quantitative assay that modifies the cDNA sequence to increase the melting temperature of short (56-64 bp) PCR amplicons, enabling their quantification in-tube by homogeneous melting-curve analysis. We used this method to analyze the expression of 8 genes, 7 potential colon cancer markers, and 1 control in samples obtained from 3 colon carcinoma cell lines, endoscopic biopsy from 8 patients undergoing gastroscopy for Barrett esophagus, and archival FFPE and frozen tissue from 20 patients who underwent surgery for colon carcinoma. RESULTS: The detection limit of the assay, when optimized for FFPE samples, was 100 copies of cDNA, and the dynamic range was 3 orders of magnitude. A prototype assay containing a panel of 8 genes displayed good reproducibility compared with the commercially available TaqMan assay (interassay CVs, 5%-20% vs 7%-43%, respectively). Gene-expression analysis was performed successfully in 26 (96%) of 27 endoscopic biopsy specimens, 30 (86%) of 35 archival FFPE samples, and 20 (100%) of 20 archival frozen samples. CONCLUSIONS: This new technology combines the reproducibility of competitive PCR with accurate quantitative detection by in-tube melting-curve analysis, enabling efficient analysis of mRNA profiles in samples with small numbers of cells or small amounts of tissue, as well as in archival FFPE tissues.

Barrett Esophagus↗