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Sample records for gene expression studies

  1. Gene expression studies using microarrays

    NARCIS (Netherlands)

    Burgess, Janette

    2001-01-01

    1. The rapid progression of the collaborative sequencing programmes that are unravelling the complete genome sequences of many organisms are opening pathways for new approaches to gene analysis. As the sequence data become available, the bottleneck in biological research will shift to understanding

  2. Studying the Complex Expression Dependences between Sets of Coexpressed Genes

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    Mario Huerta

    2014-01-01

    Full Text Available Organisms simplify the orchestration of gene expression by coregulating genes whose products function together in the cell. The use of clustering methods to obtain sets of coexpressed genes from expression arrays is very common; nevertheless there are no appropriate tools to study the expression networks among these sets of coexpressed genes. The aim of the developed tools is to allow studying the complex expression dependences that exist between sets of coexpressed genes. For this purpose, we start detecting the nonlinear expression relationships between pairs of genes, plus the coexpressed genes. Next, we form networks among sets of coexpressed genes that maintain nonlinear expression dependences between all of them. The expression relationship between the sets of coexpressed genes is defined by the expression relationship between the skeletons of these sets, where this skeleton represents the coexpressed genes with a well-defined nonlinear expression relationship with the skeleton of the other sets. As a result, we can study the nonlinear expression relationships between a target gene and other sets of coexpressed genes, or start the study from the skeleton of the sets, to study the complex relationships of activation and deactivation between the sets of coexpressed genes that carry out the different cellular processes present in the expression experiments.

  3. Serial Analysis of Gene Expression: Applications in Human Studies

    OpenAIRE

    2004-01-01

    Serial analysis of gene expression (SAGE) is a powerful tool, which provides quantitative and comprehensive expression profile of genes in a given cell population. It works by isolating short fragments of genetic information from the expressed genes that are present in the cell being studied. These short sequences, called SAGE tags, are linked together for efficient sequencing. The frequency of each SAGE tag in the cloned multimers directly reflects the transcript abundance. Therefore, SAGE r...

  4. Identification of Human HK Genes and Gene Expression Regulation Study in Cancer from Transcriptomics Data Analysis

    Science.gov (United States)

    Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun

    2013-01-01

    The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867

  5. A longitudinal study of gene expression in healthy individuals

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    Tessier Michel

    2009-06-01

    Full Text Available Abstract Background The use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22–55 years and > 55 years and gender. Methods Eleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR, as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays. Results Gene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with ~2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF, 95% of all samples profiled fell within 1.5–2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers. Conclusion This study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated

  6. A longitudinal study of gene expression in healthy individuals.

    Science.gov (United States)

    Karlovich, Chris; Duchateau-Nguyen, Guillemette; Johnson, Andrea; McLoughlin, Patricia; Navarro, Mercidita; Fleurbaey, Carole; Steiner, Lori; Tessier, Michel; Nguyen, Tracy; Wilhelm-Seiler, Monika; Caulfield, John P

    2009-06-07

    The use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22-55 years and > 55 years) and gender. Eleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays. Gene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with approximately 2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF), 95% of all samples profiled fell within 1.5-2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers. This study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated expression in a subject with iron deficiency anemia and

  7. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

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    Chapman, Joanne R; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

  8. Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

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    Turner Renee J

    2009-08-01

    Full Text Available Abstract Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT, 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder. Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

  9. Validation of housekeeping genes for studying differential gene expression in the bovine myometrium.

    Science.gov (United States)

    Rekawiecki, Robert; Kowalik, Magdalena K; Kotwica, Jan

    2013-12-01

    The aim of this study was to determine the steady-state expression of 13 selected housekeeping genes in the myometrium of cyclic and pregnant cows. Cells taken from bovine myometrium on days 1-5, 6-10, 11-16 and 17-20 of the oestrous cycle and in weeks 3-5, 6-8 and 9-12 of pregnancy were used. Reverse transcribed RNA was amplified in real-time PCR using designed primers. Reaction efficiency was determined with the Linreg programme. The geNorm and NormFinder programmes were used to select the best housekeeping genes. They calculate the expression stability factor for each used housekeeping gene with the smallest value for most stably expressed genes. According to geNorm, the most stable housekeeping genes in the myometrium were C2orf29, TPB and TUBB2B, while the least stably expressed genes were 18S RNA, HPRT1 and GAPDH. NormFinder identified the best genes in the myometrium as C2orf29, MRPL12 and TBP, while the worst genes were 18S RNA, B2M and SF3A1. Differences in stability factors between the two programmes may also indicate that the physiological status of the female, e.g. pregnancy, affects the stability of expression of housekeeping genes. The different expression stability of housekeeping genes did not affect progesterone receptor expression but it could be important if small differences in gene expression were measured between studies.

  10. Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

    Science.gov (United States)

    Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

    2015-01-01

    Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  11. Expression studies of the obesity candidate gene FTO in pig

    DEFF Research Database (Denmark)

    Madsen, Majbritt Busk; Birck, Malene Muusfeldt; Fredholm, Merete

    2010-01-01

    Obesity is an increasing problem worldwide and research on candidate genes in good animal models is highly needed. The pig is an excellent model as its metabolism, organ size, and eating habits resemble that of humans. The present study is focused on the characterization of the fat mass and obesity...... associated gene (FTO) in pig. This gene has recently been associated with increased body mass index in several human populations. To establish information on the expression profile of FTO in the pig we performed quantitative PCR in a panel of adult pig tissues and in tissues sampled at different...... and cerebellum). Additionally, in order to see the involvement of the FTO gene in obesity, the changes in expression level were investigated in a nutritional study in brain of Gottingen minipigs under a high cholesterol diet. Significantly higher (P

  12. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

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    Lucie Kosinová

    Full Text Available The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3 in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information

  13. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    Science.gov (United States)

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

  14. Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

    Directory of Open Access Journals (Sweden)

    Cora S. Thiel

    2015-01-01

    Full Text Available Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes” are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1 which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  15. Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR

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    Xiang Fengning

    2008-11-01

    Full Text Available Abstract Background The wild grass species Brachypodium distachyon (Brachypodium hereafter is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data. Results A systematic exploration of suitable reference genes in Brachypodium is presented here. Nine reference gene candidates were chosen, and their gene sequences were obtained from the Brachypodium expressed sequence tag (EST databases. Their expression levels were examined by quantitative real-time PCR (qRT-PCR using 21 different Brachypodium plant samples, including those from different plant tissues and grown under various growth conditions. Effects of plant growth hormones were also visualized in the assays. The expression stability of the candidate genes was evaluated using two analysis software packages, geNorm and NormFinder. In conclusion, the ubiquitin-conjugating enzyme 18 gene (UBC18 was validated as a suitable reference gene across all the plant samples examined. While the expression of the polyubiquitin genes (Ubi4 and Ubi10 was most stable in different plant tissues and growth hormone-treated plant samples, the expression of the S-adenosylmethionine decarboxylase gene (SamDC ranked was most stable in plants grown under various environmental stresses. Conclusion This study identified the reference genes that are most suitable for normalizing the gene expression data in Brachypodium. These reference genes will be particularly useful when stress-responsive genes are analyzed in order to produce transgenic plants that exhibit enhanced stress resistance.

  16. Optimal Design and Analysis of Genetic Studies on Gene Expression

    NARCIS (Netherlands)

    Fu, Jingyuan; Jansen, Ritsert C.

    2006-01-01

    Whole-genome profiling of gene expression in a segregating population has the potential to identify the regulatory consequences of natural allelic variation. Costs of such studies are high and require that resources—microarrays and population—are used as efficiently as possible. We show that current

  17. Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

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    Cordeiro Raposo Fernando

    2011-09-01

    Full Text Available Abstract Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H oxidoreductase; AJ457980.1, ACT2 (actin 2; TC234027, and rrn26 (a putative homologue to RNA 26S gene; AL827977.1. In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1 and TaWIN1 (14-3-3 like protein, AB042193 were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire grown under three treatments (organic, conventional and no nitrogen and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production.

  18. Paralogous Genes as a Tool to Study the Regulation of Gene Expression

    DEFF Research Database (Denmark)

    Hoffmann, Robert D

    The genomes of plants are marked by reoccurring events of whole-genome duplication. These events are major contributors to speciation and provide the genetic material for organisms to evolve ever greater complexity. Duplicated genes, referred to as paralogs, may be retained because they acquired...... new functions, or their gene products are in a dosage balance. Regulatory DNA elements - some of which are conserved across species and hence called conserved non-coding sequences (CNSs) - that control expression of duplicated genes are thus under similar purifying selection. In the present study, I...... have performed in-depth analyses of paralogous genes in Arabidopsis thaliana, their expression profile, their sequence conservation, and their functions, in order to investigate the relationship between gene expression and retention of paralogous genes. Paralogs with lower expression than...

  19. Assessment of Suitable Reference Genes for Quantitative Gene Expression Studies in Melon Fruits.

    Science.gov (United States)

    Kong, Qiusheng; Gao, Lingyun; Cao, Lei; Liu, Yue; Saba, Hameed; Huang, Yuan; Bie, Zhilong

    2016-01-01

    Melon (Cucumis melo L.) is an attractive model plant for investigating fruit development because of its morphological, physiological, and biochemical diversity. Quantification of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with stably expressed reference genes for normalization can effectively elucidate the biological functions of genes that regulate fruit development. However, the reference genes for data normalization in melon fruits have not yet been systematically validated. This study aims to assess the suitability of 20 genes for their potential use as reference genes in melon fruits. Expression variations of these genes were measured in 24 samples that represented different developmental stages of fertilized and parthenocarpic melon fruits by qRT-PCR analysis. GeNorm identified ribosomal protein L (CmRPL) and cytosolic ribosomal protein S15 (CmRPS15) as the best pair of reference genes, and as many as five genes including CmRPL, CmRPS15, TIP41-like family protein (CmTIP41), cyclophilin ROC7 (CmCYP7), and ADP ribosylation factor 1 (CmADP) were required for more reliable normalization. NormFinder ranked CmRPS15 as the best single reference gene, and RAN GTPase gene family (CmRAN) and TATA-box binding protein (CmTBP2) as the best combination of reference genes in melon fruits. Their effectiveness was further validated by parallel analyses on the activities of soluble acid invertase and sucrose phosphate synthase, and expression profiles of their respective encoding genes CmAIN2 and CmSPS1, as well as sucrose contents during melon fruit ripening. The validated reference genes will help to improve the accuracy of gene expression studies in melon fruits.

  20. Assessment of Suitable Reference Genes for Quantitative Gene Expression Studies in Melon Fruits

    Science.gov (United States)

    Kong, Qiusheng; Gao, Lingyun; Cao, Lei; Liu, Yue; Saba, Hameed; Huang, Yuan; Bie, Zhilong

    2016-01-01

    Melon (Cucumis melo L.) is an attractive model plant for investigating fruit development because of its morphological, physiological, and biochemical diversity. Quantification of gene expression by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with stably expressed reference genes for normalization can effectively elucidate the biological functions of genes that regulate fruit development. However, the reference genes for data normalization in melon fruits have not yet been systematically validated. This study aims to assess the suitability of 20 genes for their potential use as reference genes in melon fruits. Expression variations of these genes were measured in 24 samples that represented different developmental stages of fertilized and parthenocarpic melon fruits by qRT-PCR analysis. GeNorm identified ribosomal protein L (CmRPL) and cytosolic ribosomal protein S15 (CmRPS15) as the best pair of reference genes, and as many as five genes including CmRPL, CmRPS15, TIP41-like family protein (CmTIP41), cyclophilin ROC7 (CmCYP7), and ADP ribosylation factor 1 (CmADP) were required for more reliable normalization. NormFinder ranked CmRPS15 as the best single reference gene, and RAN GTPase gene family (CmRAN) and TATA-box binding protein (CmTBP2) as the best combination of reference genes in melon fruits. Their effectiveness was further validated by parallel analyses on the activities of soluble acid invertase and sucrose phosphate synthase, and expression profiles of their respective encoding genes CmAIN2 and CmSPS1, as well as sucrose contents during melon fruit ripening. The validated reference genes will help to improve the accuracy of gene expression studies in melon fruits. PMID:27536316

  1. Selection of reliable reference genes for gene expression studies in peach using real-time PCR

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    Zhou Jun

    2009-07-01

    Full Text Available Abstract Background RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch. Therefore, the present study was conducted to identify suitable reference gene(s for normalization of gene expression in peach. Results In this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT, cyclophilin (CYP2, RNA polymerase II (RP II, phospholipase A2 (PLA2, ribosomal protein L13 (RPL13, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 18S ribosomal RNA (18S rRNA, tubblin beta (TUB, tubblin alpha (TUA, translation elongation factor 2 (TEF2 and ubiquitin 10 (UBQ10. All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results. Conclusion Our study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls

  2. Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca.

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    Docimo, Teresa; Schmidt, Gregor W; Luck, Katrin; Delaney, Sven K; D'Auria, John C

    2013-01-01

    Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca ( Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.

  3. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection.

    Science.gov (United States)

    Nguyen Thi, Le Thuy; Sarmiento, Maria Elena; Calero, Romel; Camacho, Frank; Reyes, Fatima; Hossain, Md Murad; Gonzalez, Gustavo Sierra; Norazmi, Mohd Nor; Acosta, Armando

    2014-09-01

    The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.

  4. Evaluation of reference genes for gene expression studies in human brown adipose tissue.

    Science.gov (United States)

    Taube, Magdalena; Andersson-Assarsson, Johanna C; Lindberg, Kristin; Pereira, Maria J; Gäbel, Markus; Svensson, Maria K; Eriksson, Jan W; Svensson, Per-Arne

    2015-01-01

    Human brown adipose tissue (BAT) has during the last 5 year been subjected to an increasing research interest, due to its putative function as a target for future obesity treatments. The most commonly used method for molecular studies of human BAT is the quantitative polymerase chain reaction (qPCR). This method requires normalization to a reference gene (genes with uniform expression under different experimental conditions, e.g. similar expression levels between human BAT and WAT), but so far no evaluation of reference genes for human BAT has been performed. Two different microarray datasets with samples containing human BAT were used to search for genes with low variability in expression levels. Seven genes (FAM96B, GNB1, GNB2, HUWE1, PSMB2, RING1 and TPT1) identified by microarray analysis, and 8 commonly used reference genes (18S, B2M, GAPDH, LRP10, PPIA, RPLP0, UBC, and YWHAZ) were selected and further analyzed by quantitative PCR in both BAT containing perirenal adipose tissue and subcutaneous adipose tissue. Results were analyzed using 2 different algorithms (Normfinder and geNorm). Most of the commonly used reference genes displayed acceptably low variability (geNorm M-values genes identified by microarray displayed an even lower variability (M-values genes for qPCR analysis of human BAT and we recommend that they are included in future gene expression studies of human BAT.

  5. Validation of commonly used reference genes for sleep-related gene expression studies

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    Castro Rosa MRPS

    2009-05-01

    Full Text Available Abstract Background Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin, beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, and hypoxanthine guanine phosphoribosyl transferase (HPRT. Results Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF and glycerol-3-phosphate dehydrogenase1 (GPD1 was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance. Conclusion This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results.

  6. RNA preparation and characterization for gene expression studies

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2009-01-01

    Much information can be obtained from knowledge of the relative expression level of each gene in the transcriptome. With the current advances in technology as little as a single cell is required as starting material for gene expression experiments. The mRNA from a single cell may be linearly ampl...

  7. Suitability of commonly used housekeeping genes in gene expression studies for space radiation research

    Science.gov (United States)

    Arenz, A.; Stojicic, N.; Lau, P.; Hellweg, C. E.; Baumstark-Khan, C.

    Research on the effects of ionizing radiation exposure involves the use of real-time reverse transcription polymerase chain reaction (qRT-PCR) for measuring changes in gene expression. Several variables need to be controlled for gene expression analysis, such as different amounts of starting material between the samples, variations in enzymatic efficiencies of the reverse transcription step, and differences in RNA integrity. Normalization of the obtained data to an invariant endogenous control gene (reference gene) is the elementary step in relative quantification strategy. There is a strong correlation between the quality of the normalized data and the stability of the reference gene itself. This is especially relevant when the samples have been obtained after exposure to radiation qualities inducing different amounts and kinds of damage, leading to effects on cell cycle delays or even on cell cycle blocks. In order to determine suitable reference genes as internal controls in qRT-PCR assays after exposure to ionizing radiation, we studied the gene expression levels of nine commonly used reference genes which are constitutively expressed in A549 lung cancer cells. Expression levels obtained for ACTB, B2M, GAPDH, PBGD, 18S rRNA, G6PDH, HPRT, UBC, TFRC and SDHA were determined after exposure to 2 and 6 Gy X-radiation. Gene expression data for Growth arrest and damage-inducible gene 45 (GADD45α) and Cyclin-dependent kinase inhibitor 1A (CDKN1A/p21CIP1) were selected to elucidate the influence of normalization by using appropriate and inappropriate internal control genes. According to these results, we strongly recommend the use of a panel of reference genes instead of only one.

  8. Gene Expression Correlation for Cancer Diagnosis: A Pilot Study

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    Binbing Ling

    2014-01-01

    Full Text Available Poor prognosis for late-stage, high-grade, and recurrent cancers has been motivating cancer researchers to search for more efficient biomarkers to identify the onset of cancer. Recent advances in constructing and dynamically analyzing biomolecular networks for different types of cancer have provided a promising novel strategy to detect tumorigenesis and metastasis. The observation of different biomolecular networks associated with normal and cancerous states led us to hypothesize that correlations for gene expressions could serve as valid indicators of early cancer development. In this pilot study, we tested our hypothesis by examining whether the mRNA expressions of three randomly selected cancer-related genes PIK3C3, PIM3, and PTEN were correlated during cancer progression and the correlation coefficients could be used for cancer diagnosis. Strong correlations (0.68≤r≤1.0 were observed between PIK3C3 and PIM3 in breast cancer, between PIK3C3 and PTEN in breast and ovary cancers, and between PIM3 and PTEN in breast, kidney, liver, and thyroid cancers during disease progression, implicating that the correlations for cancer network gene expressions could serve as a supplement to current clinical biomarkers, such as cancer antigens, for early cancer diagnosis.

  9. Study on the Specific Gene Expression during Spermatogenesis of Rat

    Institute of Scientific and Technical Information of China (English)

    吴燕婉; 王春梅; 黄海燕; 石心泉; 邢志军; 刘德瑜

    2001-01-01

    Objective To explore specific gene expression for regulating meiosis of germ cells during spermatogenesis of rat testis Materials & Methods Male SD rats, aged 1, 3 and 8 weeks, were observed in this study. The methods of morphological observation on testicular tissues embedded by resin and mRNA differential display (DDRT-PCR) were combined to obtain specific mRNA expression gene fragments during the testicular development. Reverse dot blot hybridization was operated to further screen the positive differential DNA fragments.The positive DNA segments were sub-cloned in pGEM-T-Easy vector and transformed into the competent E-coli 109 straint. Northern blot analysis and in situ hybridization were also carried out for identifying tissue-specific expression as well as cell-specific expression DNA fragments. To screen λ-ZAP-II rat testicular gene library was searched for the original gene.Results Eighty two differential cDNA fragments were obtained through primary DDRT-PCR, among which 40 differential cDNA fragments were selected for further screening with reverse dot blot hybridization. After the reverse dot blot hybridization,12 primary differential DNA fragments were obtained. The size of DNA fragments ranged from 250 to 500 bp. The in situ hybridization of the testicular tissue showed that a specific DNA fragment derived from 8-week old rat testis, named CG14, was hybridized in adult rat testicular section, in which the positive nucleic acid signals were distributed specifically in the primary spermatocytes. Another DNA fragment derived from 1-week old rat testis, named AA 1 1, was hybridized specifically in Sertoli cell of 1-week old rat testis. Northern blot hybridization with [α-32P] dCTP labeled CG 1 4 probe, including cardiac, liver, kidney, brain, testis, and epididymis tissue mRNAs of rat, showed that an mRNA specific hybridization band, size of 1. 258 kb, was found in testis tissue and size of 1. 531 kb of another hybridization band present in epididymis

  10. A destabilized bacterial luciferase for dynamic gene expression studies

    Science.gov (United States)

    Allen, Michael S.; Wilgus, John R.; Chewning, Christopher S.; Sayler, Gary S.

    2006-01-01

    Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression. PMID:19003433

  11. Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

    Science.gov (United States)

    Guelke, Eileen; Bucan, Vesna; Liebsch, Christina; Lazaridis, Andrea; Radtke, Christine; Vogt, Peter M; Reimers, Kerstin

    2015-04-10

    For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained.

  12. Paralogous Genes as a Tool to Study the Regulation of Gene Expression

    DEFF Research Database (Denmark)

    Hoffmann, Robert D

    their duplicate were found to be under less purifying selection. A gene ontology (GO) term enrichment analysis showed that paralogs with similar expression levels were enriched in GO terms related to macromolecular complexes, whereas paralogs with different expression levels were enriched in terms associated......The genomes of plants are marked by reoccurring events of whole-genome duplication. These events are major contributors to speciation and provide the genetic material for organisms to evolve ever greater complexity. Duplicated genes, referred to as paralogs, may be retained because they acquired...... new functions, or their gene products are in a dosage balance. Regulatory DNA elements - some of which are conserved across species and hence called conserved non-coding sequences (CNSs) - that control expression of duplicated genes are thus under similar purifying selection. In the present study, I...

  13. Validation of reference genes for gene expression studies in the emerald ash borer (Agrilus planipennis)

    Institute of Scientific and Technical Information of China (English)

    Swapna Priya Rajarapu; Praveen Mamidala; Omprakash Mittapalli

    2012-01-01

    The Emerald ash borer (EAB,Agrilus planipennis Fairmaire) an exotic invasive insect pest has killed millions of ash trees (Fraxinus spp.) across North America and threatens billions more.We validated six A.planipennis reference genes (actin,ACT; beta tubulin,β- TUB; glyceraldehyde-3-phosphate dehydrogenase,GAPDH; ribosomal protein,RPL7; translation elongation factor 1α,TEF-1α; and ubiquitin,UBQ) using geNorm,Normfinder and BestKeeper for accurate determination of target messenger RNA levels in gene expression studies.The stability of the six reference genes was evaluated in different larval tissues,developmental stages and two treatments ofA.planipennis using quantitative real-time polymerase chain reaction.Although there was no consistent ranking observed among the reference genes across the samples,the overall analysis revealed TEF-1α as the most stable reference gene.GAPDH and ACT showed least stability for all the samples studied.We conclude that TEF-1α is the most appropriate reference gene for gene expression studies inA.planipennis.Results obtained can be applicable for transcript profiling in other invasive insect pests.Further,these validated reference genes could also serve as the basis for selection of candidate reference genes in any given insect system post-validation.

  14. Identification of suitable reference genes for gene expression studies of shoulder instability.

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    Mariana Ferreira Leal

    Full Text Available Shoulder instability is a common shoulder injury, and patients present with plastic deformation of the glenohumeral capsule. Gene expression analysis may be a useful tool for increasing the general understanding of capsule deformation, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR has become an effective method for such studies. Although RT-qPCR is highly sensitive and specific, it requires the use of suitable reference genes for data normalization to guarantee meaningful and reproducible results. In the present study, we evaluated the suitability of a set of reference genes using samples from the glenohumeral capsules of individuals with and without shoulder instability. We analyzed the expression of six commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, TBP and TFRC in the antero-inferior, antero-superior and posterior portions of the glenohumeral capsules of cases and controls. The stability of the candidate reference gene expression was determined using four software packages: NormFinder, geNorm, BestKeeper and DataAssist. Overall, HPRT1 was the best single reference gene, and HPRT1 and B2M composed the best pair of reference genes from different analysis groups, including simultaneous analysis of all tissue samples. GenEx software was used to identify the optimal number of reference genes to be used for normalization and demonstrated that the accumulated standard deviation resulting from the use of 2 reference genes was similar to that resulting from the use of 3 or more reference genes. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1. Although the use of different reference gene combinations yielded variable normalized quantities, the relative quantities within sample groups were similar and confirmed that no obvious differences were observed when using 2, 3 or 4 reference genes. Consequently, the use of 2 stable reference genes for normalization, especially

  15. Study of the Gene Expression Profile of Human Ovarian Carcinoma by a Gene Chip

    Institute of Scientific and Technical Information of China (English)

    Shenhua Xu; Hanzhou Mou; Chihong Zhu; Lijuan Qian; Zhengyan Yang; Ye Ying; Xianglin Liu

    2005-01-01

    OBJECTIVE To study the difference in gene expression between human ovarian carcinoma and normal ovarian tissues, and screen the novel associated genes by cDNA microarrays.METHODS Total RNA from 10 cases of ovarian cancer and from normal ovarian tissues were extracted by a single step method. The cDNA was retro-transcribed from an equal quantity of mRNA derived from the 10 cases of ovarian carcinoma and normal ovarian tissues, followed by labeling the cDNA strands with Cy5 and Cy3 fluorescence as probes. The mixed probes were hybridized with BiostarH 8464 dot human somatic cell genes.Fluorescence signals were assessed by a ScanArray 4000 laser scanner and the images analyzed by Gene Pix Pro 3.0 software with a digital computer.RESULTS By applying the cDNA microarray we found a total of 185 genes for which expression levels differed more than 5 times comparing human ovarian carcinoma with normal ovarian epithelium. Among these genes 86 were up-regulated >5 times and 99 were down regulated <0.2.CONCLUSION The cDNA microarray technique is effective in screening the differential gene expression between human ovarian cancers and normal ovarian epithelium. It is suggested that these genes identified are related to the genesis and development of ovarian carcinoma.

  16. Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don

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    Zheng Xiao

    2016-10-01

    Full Text Available The quantitative real-time polymerase chain reaction (qRT-PCR approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha, 18S (18s ribosomal RNA and RPL3 (ribosomal protein L3 were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB was the least stable. ACT5 (actin, RPL3, 18S and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase and RmPDS (phytoene dehydrogenase were assessed using EF1-α, 18S, ACT5, and RPL3 and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle.

  17. Suitability of endogenous reference genes for gene expression studies with human intraocular endothelial cells

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    Wei Ruoxin

    2013-02-01

    Full Text Available Abstract Background The use of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR has become widely applied as a method to measure transcript abundance. In order to be reflective of biological processes during health and disease this method is dependent on normalisation of data against stable endogenous controls. However, these genes can vary in their stability in different cell types. The importance of reference gene validation for a particular cell type is now well recognised and is an important step in any gene expression study. Results Cultured primary human choroidal and retinal endothelial cells were treated with the immunostimulant polyinosinic: polycytidylic acid or untreated. qRT-PCR was used to quantify the expression levels of 10 commonly used endogenous control genes, TBP, HPRT1, GAPDH, GUSB, PPIA, RPLP0, B2M, 18S rRNA, PGK1 and ACTB. Three different mathematical algorithms, GeNorm, NormFinder, and BestKeeper were used to analyse gene stability to give the most representative validation. In choroidal endothelial cells the most stable genes were ranked as HPRT1 and GUSB by GeNorm and NormFinder and HPRT1 and PPIA by BestKeeper. In retinal endothelial cells the most stable genes ranked were TBP and PGK1 by GeNorm and NormFinder and HPRT1 by BestKeeper. The least stable gene for both cell types was 18S with all 3 algorithms. Conclusions We have identified the most stable endogenous control genes in intraocular endothelial cells. It is suggested future qRT-PCR studies using these cells would benefit from adopting the genes identified in this study as the most appropriate endogenous control genes.

  18. Study of human dopamine sulfotransferases based on gene expression programming.

    Science.gov (United States)

    Si, Hongzong; Zhao, Jiangang; Cui, Lianhua; Lian, Ning; Feng, Hanlin; Duan, Yun-Bo; Hu, Zhide

    2011-09-01

    A quantitative model is developed to predict the Km of 47 human dopamine sulfotransferases by gene expression programming. Each kind of compound is represented by several calculated structural descriptors of moment of inertia A, average electrophilic reactivity index for a C atom, relative number of triple bonds, RNCG relative negative charge, HA-dependent HDSA-1, and HBCA H-bonding charged surface area. Eight fitness functions of the gene expression programming method are used to find the best nonlinear model. The best quantitative model with squared standard error and square of correlation coefficient are 0.096 and 0.91 for training data set, and 0.102 and 0.88 for test set, respectively. It is shown that the gene expression programming-predicted results with fitness function are in good agreement with experimental ones.

  19. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

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    Jakub Cieslak

    Full Text Available Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8 is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  20. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

    Science.gov (United States)

    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W

    2009-01-01

    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

  1. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    Science.gov (United States)

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.

  2. Cross-study homogeneity of psoriasis gene expression in skin across a large expression range.

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    Jeannette Bigler

    Full Text Available BACKGROUND: In psoriasis, only limited overlap between sets of genes identified as differentially expressed (psoriatic lesional vs. psoriatic non-lesional was found using statistical and fold-change cut-offs. To provide a framework for utilizing prior psoriasis data sets we sought to understand the consistency of those sets. METHODOLOGY/PRINCIPAL FINDINGS: Microarray expression profiling and qRT-PCR were used to characterize gene expression in PP and PN skin from psoriasis patients. cDNA (three new data sets and cRNA hybridization (four existing data sets data were compared using a common analysis pipeline. Agreement between data sets was assessed using varying qualitative and quantitative cut-offs to generate a DEG list in a source data set and then using other data sets to validate the list. Concordance increased from 67% across all probe sets to over 99% across more than 10,000 probe sets when statistical filters were employed. The fold-change behavior of individual genes tended to be consistent across the multiple data sets. We found that genes with 10-fold changes in either direction such as CHRM3, IL12B and IFNG. CONCLUSIONS/SIGNIFICANCE: Gene expression changes in psoriatic lesions were consistent across different studies, despite differences in patient selection, sample handling, and microarray platforms but between-study comparisons showed stronger agreement within than between platforms. We could use cut-offs as low as log10(ratio = 0.1 (fold-change = 1.26, generating larger gene lists that validate on independent data sets. The reproducibility of PP signatures across data sets suggests that different sample sets can be productively compared.

  3. In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

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    Kristiansen Glen

    2007-06-01

    Full Text Available Abstract Background Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. Results The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP and peptidylprolyl isomerase A (PPIA, all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. Conclusion Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

  4. Validation of endogenous control genes for gene expression studies on human ocular surface epithelium.

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    Bina Kulkarni

    Full Text Available PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG with quantitative reverse transcription PCR (qPCR, for identification of stably expressed endogenous control genes in the ocular surface (OS epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH, beta actin (ACTB, peptidylprolyl isomerase (PPIA, TATA-box binding protein (TBP1, hypoxanthine guanine phosphoribosyl transferase (HPRT1, beta glucuronidase (GUSB, Eucaryotic 18S ribosomal RNA (18S, phosphoglycerate kinase (PGK1, beta-2-microglobulin (B2M, ribosomal protein, large, P0 (RPLP0--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18Sstudy, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable. CONCLUSION: This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use

  5. Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca

    OpenAIRE

    2013-01-01

    Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is ...

  6. Detecting Periodic Genes from Irregularly Sampled Gene Expressions: A Comparison Study

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    Edward R. Dougherty

    2008-06-01

    Full Text Available Time series microarray measurements of gene expressions have been exploited to discover genes involved in cell cycles. Due to experimental constraints, most microarray observations are obtained through irregular sampling. In this paper three popular spectral analysis schemes, namely, Lomb-Scargle, Capon and missing-data amplitude and phase estimation (MAPES, are compared in terms of their ability and efficiency to recover periodically expressed genes. Based on in silico experiments for microarray measurements of Saccharomyces cerevisiae, Lomb-Scargle is found to be the most efficacious scheme. 149 genes are then identified to be periodically expressed in the Drosophila melanogaster data set.

  7. Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle.

    Science.gov (United States)

    Zhu, Wuzheng; Lin, Yaqiu; Liao, Honghai; Wang, Yong

    2015-01-01

    The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.

  8. Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR.

    Science.gov (United States)

    Kianianmomeni, Arash; Hallmann, Armin

    2013-12-01

    Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies.

  9. Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.

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    Thrush Anthony

    2010-01-01

    Full Text Available Abstract Background Perennial ryegrass (Lolium perenne L. is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2 were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L. samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h, a moderately, but stably expressed eEF1A (s, and combined expression of multigene eEF1A (m. NormFinder identified eEF1A (s and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples

  10. Towards a Holistic, Yet Gene-Centered Analysis of Gene Expression Profiles: A Case Study of Human Lung Cancers

    OpenAIRE

    Yuchun Guo; Eichler, Gabriel S.; Ying Feng; Ingber, Donald E.; Sui Huang

    2006-01-01

    Genome-wide gene expression profile studies encompass increasingly large number of samples, posing a challenge to their presentation and interpretation without losing the notion that each transcriptome constitutes a complex biological entity. Much like pathologists who visually analyze information-rich histological sections as a whole, we propose here an integrative approach. We use a self-organizing maps -based software, the gene expression dynamics inspector (GEDI) to analyze gene expressio...

  11. Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage

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    Gomez-Reino Juan J

    2008-01-01

    Full Text Available Abstract Background Assessment of gene expression is an important component of osteoarthritis (OA research, greatly improved by the development of quantitative real-time PCR (qPCR. This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application. Results Analyses of expression stability in cartilage from 10 patients with hip OA, 8 patients with knee OA and 10 controls without OA were done with classical statistical tests and the software programs geNorm and NormFinder. Results from the three methods of analysis were broadly concordant. Some of the commonly used reference genes, GAPDH, ACTB and 18S RNA, performed poorly in our analysis. In contrast, the rarely used TBP, RPL13A and B2M genes were the best. It was necessary to use together several of these three genes to obtain the best results. The specific combination depended, to some extent, on the type of samples being compared. Conclusion Our results provide a satisfactory set of previously unused reference genes for qPCR in hip and knee OA This confirms the need to evaluate the suitability of reference genes in every tissue and experimental situation before starting the quantitative assessment of gene expression by qPCR.

  12. Evaluation of potential reference genes for use in gene expression studies in the conifer pathogen (Heterobasidion annosum).

    Science.gov (United States)

    Raffaello, Tommaso; Asiegbu, Fred O

    2013-07-01

    The basidiomycete Heterobasidion annosum is the causative agent of butt and root rot disease of conifer trees and it's one of the most destructive conifer pathogen in the northern hemisphere. Because of the intrinsic difficulties in genome manipulation in this fungus, most studies have been focused on gene expression analysis using quantitative real time polymerase chain reaction (qPCR). qPCR is a powerful technique but its reliability resides in the correct selection of a set of reference genes used in the data normalization. In this study, we determined the expression stability of 11 selected reference genes in H. annosum. Almost nothing has so far been published about validation of a set of reference genes to be used in gene expression experiments in this fungus. Eleven reference genes were validated in H. annosum which was grown on three different substrates: pine bark, pine heartwood, and pine sapwood. Bestkeeper and NormFinder Excel-based software were used to evaluate the reference gene transcripts' stability. The results from these two programs indicated that three reference genes namely Tryp metab, RNA Pol3 TF, and Actin were stable in H. annosum in the conditions studied. Interestingly, the GAPDH transcript which has been extensively used in qPCR data normalization is not the best choice when a wide reference gene selection is available. This work represents the first extensive validation of reference genes in H. annosum providing support for gene expression studies and benefits for the wider forest pathology community.

  13. Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    徐永忠; 谢建平; 李瑶; 乐军; 陈建平; 淳于利娟; 王洪海

    2003-01-01

    Objective To examine the global effects of Mycobacterium tuberculosis (M.tuberculosis) infection on macrophages. Methods The gene expression profiling of macrophage U937, in response to infection with M.tuberculosis H37Ra, was monitored using a high-density cDNA microarray. Results M.tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M.tuberculosis infection and intracellular survival. Conclusions M.tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M.tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

  14. Reference genes for quantitative gene expression studies in multiple avian species.

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    Philipp Olias

    Full Text Available Quantitative real-time PCR (qPCR rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ on different tissues of the mallard (Anas platyrhynchos, domestic chicken (Gallus gallus domesticus, common crane (Grus grus, white-tailed eagle (Haliaeetus albicilla, domestic turkey (Meleagris gallopavo f. domestica, cockatiel (Nymphicus hollandicus, Humboldt penguin (Sphenicus humboldti, ostrich (Struthio camelus and zebra finch (Taeniopygia guttata, spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

  15. Reference genes for quantitative gene expression studies in multiple avian species.

    Science.gov (United States)

    Olias, Philipp; Adam, Iris; Meyer, Anne; Scharff, Constance; Gruber, Achim D

    2014-01-01

    Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

  16. Identification of stable reference genes for gene expression studies using quantitative real time PCR in buffalo oocytes and embryos.

    Science.gov (United States)

    Kumar, Parveen; Yadav, Poonam; Verma, Arpana; Singh, Dheer; De, Sachinandan; Datta, Tirtha Kumar

    2012-12-01

    The present study was aimed to validate expression stability of 6 housekeeping genes (viz. YWHAZ, SDHA, GAPDH, RPS15, RPS18 and RN18S1) in the oocytes and embryos of different stages in buffalo. A modified Trizol protocol was optimized for RNA isolation from as few as five oocytes. The expression level of selected genes was studied by an optimized real time PCR using DCT method and their stability of expression was evaluated by Microsoft Excel based visual application, geNORM. The analysis revealed that the RPS15 and GAPDH were the most stable genes across different samples. Also, the geometric mean of three genes (i.e. RPS15,RPS18 and GAPDH) were found suitable for normalization of real time PCR data from buffalo oocytes⁄embryos. The information would help in more accurate interpretation of gene expression data from oocytes⁄embryos towards understanding the molecular events in these cells during development.

  17. Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR

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    Reith Michael

    2008-03-01

    Full Text Available Abstract Background Flatfish metamorphosis involves major physiological and morphological changes. Due to its importance in aquaculture and as a model for developmental studies, some gene expression studies have focused on the understanding of this process using quantitative real-time PCR (qRT-PCR technique. Therefore, adequate reference genes for accurate normalization are required. Results The stability of 12 potential reference genes was examined during larval development in Senegalese sole (Solea senegalensis and Atlantic halibut (Hippoglossus hippoglossus to determine the most suitable genes for qRT-PCR analysis. Transcription levels of genes encoding β-Actin (ACTB, glyceraldehyde-3P-dehydrogenase (GAPDH, annexin A2 (ANXA2, glutathione S-transferase (GST, ornithine decarboxylase (ODC, hypoxanthine phosphoribosyltransferase (HPRT1, ubiquitin (UBQ, elongation factor 1 alpha (eEF1A1, 18S ribosomal RNA, and the ribosomal proteins S4 (RPS4 and L13a (RPL13a were quantitated. Two paralogous genes for ACTB were analyzed in each of both flatfish species. In addition, two paralogous genes for GAPDH were studied in Senegalese sole. RPL13a represented non-orthologous genes between both flatfish species. GeNorm and NormFinder analyses for expression stability revealed RPS4, UBQ and eEF1A1 as the most stable genes in Senegalese sole, Atlantic halibut and in a combined analysis. In all cases, paralogous genes exhibited differences in expression stability. Conclusion This work suggests RPS4, UBQ, and eEF1A1 genes as useful reference genes for accurate normalization in qRT-PCR studies in Senegalese sole and Atlantic halibut larvae. The congruent results between both species in spite of the drastic differences in larval development suggest that selected housekeeping genes (HKGs could be useful in other flatfish species. However, the finding of paralogous gene copies differentially expressed during development in some HKGs underscores the necessity to

  18. Reference genes for quantitative gene expression studies in multiple avian species

    DEFF Research Database (Denmark)

    Olias, Philipp; Adam, Iris; Meyer, Anne S.

    2014-01-01

    reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different......Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian...... species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM...

  19. Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

    Science.gov (United States)

    Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

    2016-08-01

    Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

  20. Studies of Differentially-Expressed Genes in Human Endometrial Cancer of Various Differentiated Grades

    Institute of Scientific and Technical Information of China (English)

    Bin Cai; David Hogg; Guangzhong Lu; Ling Liu; Xiaowei Xi; Wei Xu; Huifang Lu; Yongbin Yang; Xiaoping Wan

    2007-01-01

    OBJECTIVE To study the gene expression profiles of human endometrial cancers at various differentiaOted grade levels and to identify the genes related to differentiation of the endometrial cancers. METHODS cDNA microarray technology was used to analyze the differentially-expressed genes among different differentiated grades of 32 cases of endometrial cancer. Hierarchical cluster analysis (HCA) for the gene expression profiles of the cases was employed. RESULTS The tissue samples were grouped based on the various dif ferentiated tumor grades with 33 differentiation-related genes identified out (P<0.001). Based on the results from the HCA, the conformity rate was 91% among the 33 differentially-expressed genes and the analysis of pathological classification.CONCLUSION Genes related to the differentiation of endometrial cancer can be identified by using gene chips to analyze the expression profiles of endometrial cancers at various differentiated grades; HCA of the gene expression profiles can be helpful for distinguishing high-risk endometrial cancers before surgery.

  1. A combined analysis of microarray gene expression studies of the human prefrontal cortex identifies genes implicated in schizophrenia.

    Science.gov (United States)

    Pérez-Santiago, Josué; Diez-Alarcia, Rebeca; Callado, Luis F; Zhang, Jin X; Chana, Gursharan; White, Cory H; Glatt, Stephen J; Tsuang, Ming T; Everall, Ian P; Meana, J Javier; Woelk, Christopher H

    2012-11-01

    Small cohort sizes and modest levels of gene expression changes in brain tissue have plagued the statistical approaches employed in microarray studies investigating the mechanism of schizophrenia. To combat these problems a combined analysis of six prior microarray studies was performed to facilitate the robust statistical analysis of gene expression data from the dorsolateral prefrontal cortex of 107 patients with schizophrenia and 118 healthy subjects. Multivariate permutation tests identified 144 genes that were differentially expressed between schizophrenia and control groups. Seventy of these genes were identified as differentially expressed in at least one component microarray study but none of these individual studies had the power to identify the remaining 74 genes, demonstrating the utility of a combined approach. Gene ontology terms and biological pathways that were significantly enriched for differentially expressed genes were related to neuronal cell-cell signaling, mesenchymal induction, and mitogen-activated protein kinase signaling, which have all previously been associated with the etiopathogenesis of schizophrenia. The differential expression of BAG3, C4B, EGR1, MT1X, NEUROD6, SST and S100A8 was confirmed by real-time quantitative PCR in an independent cohort using postmortem human prefrontal cortex samples. Comparison of gene expression between schizophrenic subjects with and without detectable levels of antipsychotics in their blood suggests that the modulation of MT1X and S100A8 may be the result of drug exposure. In conclusion, this combined analysis has resulted in a statistically robust identification of genes whose dysregulation may contribute to the mechanism of schizophrenia.

  2. Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum.

    Science.gov (United States)

    Xu, Zhichao; Xu, Jiang; Ji, Aijia; Zhu, Yingjie; Zhang, Xin; Hu, Yuanlei; Song, Jingyuan; Chen, Shilin

    2015-12-15

    Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for the accurate analysis of gene expression. However, high homology among gene families might result in unsuitability of reference genes, which leads to the inaccuracy of qRT-PCR analysis. The release of the Ganoderma lucidum genome has triggered numerous studies to be done on the homology among gene families with the purpose of selecting reliable reference genes. Based on the G. lucdum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization. The result indicated that commonly used polyubiquitin (PUB), beta-actin (BAT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unsuitable reference genes because of the high sequence similarity and low primer specificity. According to the evaluation of RefFinder, cyclophilin 5 (CYP5) was ranked as the most stable reference gene for 27 tested samples under all experimental conditions and eighteen mycelial samples. Based on sequence analysis and expression analysis, our study suggested that gene characteristic, primer specificity of high homologous genes, allele-specificity expression of candidate genes and under-evaluation of reference genes influenced the accuracy and sensitivity of qRT-PCR analysis. This investigation not only revealed potential factors influencing the unsuitability of reference genes but also selected the superior reference genes from more candidate genes and testing samples than those used in the previous study. Furthermore, our study established a model for reference gene analysis by using the genomic sequence.

  3. Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model.

    Science.gov (United States)

    Pereira-Fantini, Prue M; Rajapaksa, Anushi E; Oakley, Regina; Tingay, David G

    2016-05-23

    Preterm newborns often require invasive support, however even brief periods of supported ventilation applied inappropriately to the lung can cause injury. Real-time quantitative reverse transcriptase-PCR (qPCR) has been extensively employed in studies of ventilation-induced lung injury with the reference gene 18S ribosomal RNA (18S RNA) most commonly employed as the internal control reference gene. Whilst the results of these studies depend on the stability of the reference gene employed, the use of 18S RNA has not been validated. In this study the expression profile of five candidate reference genes (18S RNA, ACTB, GAPDH, TOP1 and RPS29) in two geographical locations, was evaluated by dedicated algorithms, including geNorm, Normfinder, Bestkeeper and ΔCt method and the overall stability of these candidate genes determined (RefFinder). Secondary studies examined the influence of reference gene choice on the relative expression of two well-validated lung injury markers; EGR1 and IL1B. In the setting of the preterm lamb model of lung injury, RPS29 reference gene expression was influenced by tissue location; however we determined that individual ventilation strategies influence reference gene stability. Whilst 18S RNA is the most commonly employed reference gene in preterm lamb lung studies, our results suggest that GAPDH is a more suitable candidate.

  4. Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA

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    Cinar Mehmet Ulas

    2013-02-01

    Full Text Available Abstract Background As an in vitro model porcine peripheral blood mononuclear cells (PBMCs is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs. Results The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P  Conclusion There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder. In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram

  5. Study of integrated heterogeneous data reveals prognostic power of gene expression for breast cancer survival.

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    Richard E Neapolitan

    Full Text Available Studies show that thousands of genes are associated with prognosis of breast cancer. Towards utilizing available genetic data, efforts have been made to predict outcomes using gene expression data, and a number of commercial products have been developed. These products have the following shortcomings: 1 They use the Cox model for prediction. However, the RSF model has been shown to significantly outperform the Cox model. 2 Testing was not done to see if a complete set of clinical predictors could predict as well as the gene expression signatures.We address these shortcomings. The METABRIC data set concerns 1981 breast cancer tumors. Features include 21 clinical features, expression levels for 16,384 genes, and survival. We compare the survival prediction performance of the Cox model and the RSF model using the clinical data and the gene expression data to their performance using only the clinical data. We obtain significantly better results when we used both clinical data and gene expression data for 5 year, 10 year, and 15 year survival prediction. When we replace the gene expression data by PAM50 subtype, our results are significant only for 5 year and 15 year prediction. We obtain significantly better results using the RSF model over the Cox model. Finally, our results indicate that gene expression data alone may predict long-term survival.Our results indicate that we can obtain improved survival prediction using clinical data and gene expression data compared to prediction using only clinical data. We further conclude that we can obtain improved survival prediction using the RSF model instead of the Cox model. These results are significant because by incorporating more gene expression data with clinical features and using the RSF model, we could develop decision support systems that better utilize heterogeneous information to improve outcome prediction and decision making.

  6. Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

    Directory of Open Access Journals (Sweden)

    Lee Yeon-Su

    2010-05-01

    Full Text Available Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. Methods We assessed the suitability of six possible reference genes, beta-actin (ACTB, glyceraldehydes-3-phosphate dehydrogenase (GAPDH, hypoxanthine phosphoribosyl transferase 1 (HPRT1, beta-2-microglobulin (B2M, ribosomal subunit L29 (RPL29 and 18S ribosomal RNA (18S rRNA in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. Results This RT-qPCR study showed that there are statistically significant (p Conclusion This study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.

  7. Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

    Directory of Open Access Journals (Sweden)

    Levy Finn Olav

    2010-03-01

    Full Text Available Abstract Background Quantitative real-time RT-PCR (RT-qPCR is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s. In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI heart failure and across developmental stages in fetal and neonatal rat myocardium. Results The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions. Conclusions The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy. The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

  8. Are Gene Expression Microarray Analyses Reliable? A Review of Studies of Retinoic Acid Responsive Genes

    Institute of Scientific and Technical Information of China (English)

    PeterJ.vanderSpek; AndreasKremer; LynnMurry; MichaelG.Walker

    2003-01-01

    Microarray analyses of gene expression are widely used,but reports of the same analyses by different groups give widely divergent results,and raise questions regarding reproducibility and reliability.We take as an example recent published reports on microarray experiments that were designed to identify retinoic acid responsive genes.These reports show substantial differences in their results.In this article,we review the methodology,results,and potential causes of differences in these applications of microarrays.Finally,we suggest practices to improve the reliability and reproducibility of microarray experiments.

  9. Are Gene Expression Microarray Analyses Reliable? A Review of Studies of Retinoic Acid Responsive Genes

    Institute of Scientific and Technical Information of China (English)

    Peter J. van der Spek; Andreas Kremer; Lynn Murry; Michael G. Walker

    2003-01-01

    Microarray analyses of gene expression are widely used, but reports of the same analyses by different groups give widely divergent results, and raise questions regarding reproducibility and reliability. We take as an example recent published reports on microarray experiments that were designed to identify retinoic acid responsive genes. These reports show substantial differences in their results. In this article, we review the methodology, results, and potential causes of differences in these applications of microarrays. Finally, we suggest practices to improve the reliability and reproducibility of microarray experiments.

  10. Integrating murine gene expression studies to understand obstructive lung disease due to chronic inhaled endotoxin.

    Directory of Open Access Journals (Sweden)

    Peggy S Lai

    Full Text Available RATIONALE: Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD. These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease. METHODS: We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke. RESULTS: A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature. CONCLUSIONS: Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene

  11. Technical considerations in the use of 18s rRNA in gene expression studies

    Science.gov (United States)

    Gene expression analysis is now commonly used in ecotoxicological studies to indicate exposure of an organism to xenobiotics. For example, the vitellogenin gene is used to diagnose exposure of fish to environmental estrogens. Reverse transcription polymerase chain reaction (RT-PC...

  12. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Directory of Open Access Journals (Sweden)

    Jing Li

    Full Text Available Antisense and RNA interference (RNAi-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  13. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Science.gov (United States)

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-03-03

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  14. Molecular mechanisms in muscular dystrophy: a gene expression profiling study.

    OpenAIRE

    2006-01-01

    The muscular dystrophies are a group of neuromuscular disorders characterized by progres¬sive muscle weakness and wasting. Although the underlying genetic defects of a large number of muscular dystrophies are now know, the molecular mechanisms resulting in the devastating effects of the disease are not yet clear. Furthermore, the muscular dystrophies differ in clinical presentation and severity. The processes responsible for this di¬vergence are largely unknown as well. In this thesis, gene e...

  15. Field study of cyclic hypoxic effects on gene expression in grass shrimp hepatopancreas.

    Science.gov (United States)

    Li, Tiandao; Brouwer, Marius

    2013-12-01

    Grass shrimp, Palaemonetes pugio, are widely used for ecological and toxicological research. They commonly experience cyclic hypoxia in their natural habitats. The response of grass shrimp to laboratory-controlled cyclic hypoxia has been studied in detail, but little is known about how field acclimatized grass shrimp regulate the gene expression and response to cyclic hypoxia. In this study we examined morphometric parameters, relative fecundity and gene expression of grass shrimp collected from two areas in Weeks Bay (Mobile, Alabama). One is a traditionally normoxic location (WBM), and the other is a traditionally cyclic hypoxic location (WC). In the week preceding grass shrimp collection dissolved oxygen (DO) at the field sites was measured continuously. DO was shrimp were significantly greater than weight and length of WC shrimp. WBM shrimp had more eggs than WC shrimp, but the difference was not significant. Shrimp from WC had a significant higher number of parasites than those from WBM. A cDNA microarray was utilized to investigate the changes in gene expression in grass shrimp hepatopancreas. Five genes, previously identified as hypoxia/cyclic hypoxia-responsive genes in laboratory exposure studies, were significantly up-regulated in WC shrimp relative to WBM. A total of 5 genes were significantly down-regulated in the field study. Only one of those genes, vitellogenin, has been previously found in chronic and cyclic hypoxic studies. Up and down-regulation of 7 selected genes was confirmed by qPCR. The overall pattern of gene expression in wild shrimp from cyclic DO sites in Weeks Bay showed only weak correlations with gene expression in shrimp from chronic and cyclic hypoxic laboratory studies. It appears therefore that transcriptome profiles of laboratory acclimated animals are of limited utility for understanding responses in field acclimatized animals that are exposed to a broader array of environmental variables.

  16. Impact of cigarette smoke on the human and mouse lungs: a gene-expression comparison study.

    Directory of Open Access Journals (Sweden)

    Mathieu C Morissette

    Full Text Available Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases.

  17. Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus.

    Science.gov (United States)

    Hibbeler, Sascha; Scharsack, Joern P; Becker, Sven

    2008-01-29

    During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG), depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. In the three-spined stickleback, Gasterosteus aculeatus, other HKG than the one for beta-actin have not been established so far. To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT) PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and NormFinder. Our results showed that in most of the tissues and treatments HKG that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin. As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

  18. Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

    Directory of Open Access Journals (Sweden)

    Becker Sven

    2008-01-01

    Full Text Available Abstract Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG, depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. In the three-spined stickleback, Gasterosteus aculeatus, other HKG than the one for beta-actin have not been established so far. Results To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and NormFinder. Our results showed that in most of the tissues and treatments HKG that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin. Conclusion As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

  19. Facts and artifacts in studies of gene expression in aneuploids and sex chromosomes.

    Science.gov (United States)

    Birchler, James A

    2014-10-01

    Studies of gene expression in aneuploids have often made the assumption that measurements of RNA abundance from the varied chromosome will establish whether there is a dosage effect or compensation. Typical procedures of RNA isolation and use of equal amounts of RNA for quantitative estimates will not measure the total transcriptome size nor the absolute expression levels per cell. Use of internal endogenous standards or averages from unvaried chromosomes for normalizations makes the assumption that there are no global modulations across the genome. However, studies that use controls to test these assumptions reveal that there are in fact often modulations on all chromosomes. The same caveats apply to gene expression studies of sex chromosomes, which also involve changes in dosage of a small portion of the genome. Here, we describe some of the pitfalls of studies of aneuploidy and sex chromosome gene expression and review methods that have been used to avoid them.

  20. [Conflict: induction-inhibition of transgene bacteria luminescence in studying expression of lux-genes].

    Science.gov (United States)

    Lesniak, D V; Popova, L Iu

    2002-01-01

    The relationship between the induction of the luminescent operon of lux-genes fused with the naphthalene and salicylate degradation genes and the inhibition of light emission caused by these compounds was studied. The quantitative correlations between these processes manifest themselves in the fact that light intensity linearly increased in a narrow concentration range of the inductor and then decreased due to the inhibition of the luminescence reaction itself, which is not related to the regulation of expression of lux-genes.

  1. Influence of the experimental design of gene expression studies on the inference of gene regulatory networks: environmental factors

    Directory of Open Access Journals (Sweden)

    Frank Emmert-Streib

    2013-02-01

    Full Text Available The inference of gene regulatory networks gained within recent years a considerable interest in the biology and biomedical community. The purpose of this paper is to investigate the influence that environmental conditions can exhibit on the inference performance of network inference algorithms. Specifically, we study five network inference methods, Aracne, BC3NET, CLR, C3NET and MRNET, and compare the results for three different conditions: (I observational gene expression data: normal environmental condition, (II interventional gene expression data: growth in rich media, (III interventional gene expression data: normal environmental condition interrupted by a positive spike-in stimulation. Overall, we find that different statistical inference methods lead to comparable, but condition-specific results. Further, our results suggest that non-steady-state data enhance the inferability of regulatory networks.

  2. Identification and validation of reference genes for gene expression studies in sweet osmanthus (Osmanthus fragrans) based on transcriptomic sequence data

    Indian Academy of Sciences (India)

    HONGNA MU; TAOZE SUN; CHEN XU; LIANGGUI WANG; YUANZHENG YUE; HUOGEN LI; XIULIAN YANG

    2017-06-01

    Accurate normalized data is a primary requisite for quantifying gene expression using RT-qPCR technology. Despite this importance, however, suitable reference genes in Osmanthus fragrans are not available. In this study, seven potentialcandidate reference genes (OfL25-1, OfL25-10, OfRP2, OfTUA, OfTUB3, OfUBQ2 and Of18S) were evaluated to determine which one would be the most reliable reference genes. The expression levels of the candidate referencegenes were analysed by RT-qPCR in flower, leaf, pedicel, blossom bud tissues, as well as in floral organs at different developmental stages.GeNormand NormFinderwere used to statistically analyse transcript variation.Results indicated that OfRP2 and OfL25-10 were the optimal reference genes for use in RT-qPCR when analysing different stages of floral development; while OfTUB3 and OfL25-1 were optimal across tissues. The selected reference genes were used to examineOfMYB1 expression. The results appeared to be useful for future gene expression analyses aiming to characterize developmental stages and tissues of O. fragrans.

  3. Genes and gene expression: Localization, damage and control -- A multilevel and inter-disciplinary study

    Energy Technology Data Exchange (ETDEWEB)

    Ts' o, P.O.P.

    1990-09-01

    All projects are working toward a goal for describing the three dimensional nuclear topography in terms of relative spatial relationships among genes (specific DNA sequence). Methods are now being perfected to detect these genes, quantitatively and spatially, to perturb these genes specifically, and to measure the perturbation in order to assure specificity. We are developing methods to assay, after perturbation of the target DNA within living cells, whether or not only the target sequence are attacked while other sequences remain unharmed. We are now at the stage to do chemical gene modification or masking within living cells in a strictly sequence-specific manner. Soon, we will be able to study the function and the physical location of each gene in living cells with exquisite specificity. 25 refs., 15 figs.

  4. [Comparative study of therapy targeted genes expression in neuroblastoma cell lines].

    Science.gov (United States)

    Lebedev, T D; Spirin, P V; Orlova, N N; Prokofjeva, M M; Prassolov, V S

    2015-01-01

    In this study we evaluated c-kit, VEGFA, and MYC gene expression level in seven neuroblastoma stable cell lines: SK-N-SH, SK-N-BE, SK-N-AS, SH-SY5Y, Kelly, IMR-32, and LAN-1. Expression levels of these genes can serve as diagnostic factors of cancer progression, and proteins encoded by these genes are promising targets for neuroblastoma treatment. SH-SY5Y and SK-N-AS cells have highest MYC expression and the same VEGFA expression, although SH-SY5Y has 10 times higher c-kit expression than SK-N-AS cells. Both IMR-32 and LAN-1 cells have low MYC expression level, but differ in c-kit expression, IMR-32 has significantly higher c-kit expression, than any other neuroblastoma cell line. LAN-1 on the other hand has the highest VEGFA expression. These data suggest that MYC, c-kit, and VEGFA genes can play different roles in development and progression of neuroblastoma depending on other activated molecular mechanisms in malignant cells.

  5. A Comparative Study of Feature Selection and Classification Methods for Gene Expression Data

    KAUST Repository

    Abusamra, Heba

    2013-05-01

    Microarray technology has enriched the study of gene expression in such a way that scientists are now able to measure the expression levels of thousands of genes in a single experiment. Microarray gene expression data gained great importance in recent years due to its role in disease diagnoses and prognoses which help to choose the appropriate treatment plan for patients. This technology has shifted a new era in molecular classification, interpreting gene expression data remains a difficult problem and an active research area due to their native nature of “high dimensional low sample size”. Such problems pose great challenges to existing classification methods. Thus, effective feature selection techniques are often needed in this case to aid to correctly classify different tumor types and consequently lead to a better understanding of genetic signatures as well as improve treatment strategies. This thesis aims on a comparative study of state-of-the-art feature selection methods, classification methods, and the combination of them, based on gene expression data. We compared the efficiency of three different classification methods including: support vector machines, k- nearest neighbor and random forest, and eight different feature selection methods, including: information gain, twoing rule, sum minority, max minority, gini index, sum of variances, t- statistics, and one-dimension support vector machine. Five-fold cross validation was used to evaluate the classification performance. Two publicly available gene expression data sets of glioma were used for this study. Different experiments have been applied to compare the performance of the classification methods with and without performing feature selection. Results revealed the important role of feature selection in classifying gene expression data. By performing feature selection, the classification accuracy can be significantly boosted by using a small number of genes. The relationship of features selected in

  6. Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.).

    Science.gov (United States)

    Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

    2016-01-01

    The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.

  7. Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L..

    Directory of Open Access Journals (Sweden)

    Maryam Moazzam Jazi

    Full Text Available The tree species, Pistacia vera (P. vera is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.

  8. Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA

    Directory of Open Access Journals (Sweden)

    Cinar Mehmet

    2012-02-01

    Full Text Available Abstract Background To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs. In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs lipopolysaccharide (LPS and lipoteichoic acid (LTA. Three different algorithms (geNorm, Normfinder and BestKeeper were applied to assess the stability of HKGs. Results The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ was determined by qRT-PCR in AMs that were stimulated by LPS and LTA in vitro. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P SDHA, B2M and RPL4 showed a high expression stability in the irrespective to the stimulation group, while SDHA, YWHAZ and RPL4 showed high stability in non-stimulated control group. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that SDHA was the most stable gene in all the groups. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study. Conclusions There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of

  9. Systematic enrichment analysis of gene expression profiling studies identifies consensus pathways implicated in colorectal cancer development

    Directory of Open Access Journals (Sweden)

    Jesús Lascorz

    2011-01-01

    Full Text Available Background: A large number of gene expression profiling (GEP studies on colorectal carcinogenesis have been performed but no reliable gene signature has been identified so far due to the lack of reproducibility in the reported genes. There is growing evidence that functionally related genes, rather than individual genes, contribute to the etiology of complex traits. We used, as a novel approach, pathway enrichment tools to define functionally related genes that are consistently up- or down-regulated in colorectal carcinogenesis. Materials and Methods: We started the analysis with 242 unique annotated genes that had been reported by any of three recent meta-analyses covering GEP studies on genes differentially expressed in carcinoma vs normal mucosa. Most of these genes (218, 91.9% had been reported in at least three GEP studies. These 242 genes were submitted to bioinformatic analysis using a total of nine tools to detect enrichment of Gene Ontology (GO categories or Kyoto Encyclopedia of Genes and Genomes (KEGG pathways. As a final consistency criterion the pathway categories had to be enriched by several tools to be taken into consideration. Results: Our pathway-based enrichment analysis identified the categories of ribosomal protein constituents, extracellular matrix receptor interaction, carbonic anhydrase isozymes, and a general category related to inflammation and cellular response as significantly and consistently overrepresented entities. Conclusions: We triaged the genes covered by the published GEP literature on colorectal carcinogenesis and subjected them to multiple enrichment tools in order to identify the consistently enriched gene categories. These turned out to have known functional relationships to cancer development and thus deserve further investigation.

  10. [Study of testicular cancer gene expression in samples of oral leukoplakia and squamous cell carcinoma of the mouth].

    Science.gov (United States)

    Skorodumova, L O; Muraev, A A; Zakharova, E S; Shepelev, M V; Korobko, I V; Zaderenko, I A; Ivanov, S Iu; Gnuchev, N V; Georgiev, G P; Larin, S S

    2012-01-01

    Cancer-testis (CT) antigens are normally expressed mostly in human germ cells, there is also an aberrant expression in some tumor cells. This expression profile makes them potential tumor growth biomarkers and a promising target for tumor immunotherapy. Specificity of CT genes expression in oral malignant and potentially malignant diseases, e.g. oral leukoplakia, is not yet studied. Data on CT genes expression profile in leukoplakia would allow developing new diagnostic methods with potential value for immunotherapy and prophylaxis of leukoplakia malignization. In our study we compared CT genes expression in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma. We are the first to describe CT genes expression in oral leukoplakia without dysplasia. This findings make impossible differential diagnosis of oral leukoplakia and squamous cell carcinoma on the basis of CT genes expression. The prognostic value of CT genes expression is still unclear, therefore the longitudinal studies are necessary.

  11. Study with microarrays of the differential gene expression profiles of glioblastoma

    Institute of Scientific and Technical Information of China (English)

    YANG Zhi-lin; XU Ru-xiang; JIANG Xiao-dan; KE Yi-quan; LUO Cheng-yi; JIN Ying; HU Gen-xi

    2001-01-01

    Objective: This study aims to screen the differentially expressed genes of glioblastoma using microarray technique. Methods: Specimens of glioblastoma and normal brain tissue were obtained from pathologically confirmed patients.A cDNA microarray comprising 14 000 clones covering the whole sets of the retro-transcriptional products of the mRNAs of various gliomas and those of normal brain tissues was established, with which the differences in gene expression between glioblastoma and normal brain tissues were investigated. Results: It was found that 94 genes were more than 3-fold differentially expressed with 298 more than doubled in the glioblastoma in comparison with the normal brain tissue. Some over-expressed genes in the glioblastoma were scarcely expressed in normal brain tissues, and several novel genes that may have biological relevance in the process ofglioma genesis were identified. Conclusion: Microarray technique combined with relevant cDNA repository can facilitate rapid large-scale identification of potential target genes for diagnosis and.therapy of glioma.

  12. Study of liver function and expression of some detoxification genes ...

    African Journals Online (AJOL)

    Ahmad Ali Badr

    2015-10-19

    Oct 19, 2015 ... stress. Study of alteration of end organ markers and mRNA due to MTBE ... Results: Statistical analysis showed that in the MTBE treated groups, serum albumin ... Production and hosting by Elsevier B.V. on behalf of Ain Shams University. ... MTBE is rapidly and readily absorbed via inhalation and oral.

  13. An adaptively weighted statistic for detecting differential gene expression when combining multiple transcriptomic studies

    OpenAIRE

    Li, Jia; Tseng, George C

    2011-01-01

    Global expression analyses using microarray technologies are becoming more common in genomic research, therefore, new statistical challenges associated with combining information from multiple studies must be addressed. In this paper we will describe our proposal for an adaptively weighted (AW) statistic to combine multiple genomic studies for detecting differentially expressed genes. We will also present our results from comparisons of our proposed AW statistic to Fisher...

  14. Evaluation of phenoxybenzamine in the CFA model of pain following gene expression studies and connectivity mapping.

    Science.gov (United States)

    Chang, Meiping; Smith, Sarah; Thorpe, Andrew; Barratt, Michael J; Karim, Farzana

    2010-09-16

    We have previously used the rat 4 day Complete Freund's Adjuvant (CFA) model to screen compounds with potential to reduce osteoarthritic pain. The aim of this study was to identify genes altered in this model of osteoarthritic pain and use this information to infer analgesic potential of compounds based on their own gene expression profiles using the Connectivity Map approach. Using microarrays, we identified differentially expressed genes in L4 and L5 dorsal root ganglia (DRG) from rats that had received intraplantar CFA for 4 days compared to matched, untreated control animals. Analysis of these data indicated that the two groups were distinguishable by differences in genes important in immune responses, nerve growth and regeneration. This list of differentially expressed genes defined a "CFA signature". We used the Connectivity Map approach to identify pharmacologic agents in the Broad Institute Build02 database that had gene expression signatures that were inversely related ('negatively connected') with our CFA signature. To test the predictive nature of the Connectivity Map methodology, we tested phenoxybenzamine (an alpha adrenergic receptor antagonist) - one of the most negatively connected compounds identified in this database - for analgesic activity in the CFA model. Our results indicate that at 10 mg/kg, phenoxybenzamine demonstrated analgesia comparable to that of Naproxen in this model. Evaluation of phenoxybenzamine-induced analgesia in the current study lends support to the utility of the Connectivity Map approach for identifying compounds with analgesic properties in the CFA model.

  15. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

    Science.gov (United States)

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-01

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  16. Gene Expression-Based Survival Prediction in Lung Adenocarcinoma: A Multi-Site, Blinded Validation Study

    Science.gov (United States)

    Shedden, Kerby; Taylor, Jeremy M.G.; Enkemann, Steve A.; Tsao, Ming S.; Yeatman, Timothy J.; Gerald, William L.; Eschrich, Steve; Jurisica, Igor; Venkatraman, Seshan E.; Meyerson, Matthew; Kuick, Rork; Dobbin, Kevin K.; Lively, Tracy; Jacobson, James W.; Beer, David G.; Giordano, Thomas J.; Misek, David E.; Chang, Andrew C.; Zhu, Chang Qi; Strumpf, Dan; Hanash, Samir; Shepherd, Francis A.; Ding, Kuyue; Seymour, Lesley; Naoki, Katsuhiko; Pennell, Nathan; Weir, Barbara; Verhaak, Roel; Ladd-Acosta, Christine; Golub, Todd; Gruidl, Mike; Szoke, Janos; Zakowski, Maureen; Rusch, Valerie; Kris, Mark; Viale, Agnes; Motoi, Noriko; Travis, William; Sharma, Anupama

    2009-01-01

    Although prognostic gene expression signatures for survival in early stage lung cancer have been proposed, for clinical application it is critical to establish their performance across different subject populations and in different laboratories. Here we report a large, training-testing, multi-site blinded validation study to characterize the performance of several prognostic models based on gene expression for 442 lung adenocarcinomas. The hypotheses proposed examined whether microarray measurements of gene expression either alone or combined with basic clinical covariates (stage, age, sex) can be used to predict overall survival in lung cancer subjects. Several models examined produced risk scores that substantially correlated with actual subject outcome. Most methods performed better with clinical data, supporting the combined use of clinical and molecular information when building prognostic models for early stage lung cancer. This study also provides the largest available set of microarray data with extensive pathological and clinical annotation for lung adenocarcinomas. PMID:18641660

  17. Functional organisation of central cardiovascular pathways: studies using c-fos gene expression.

    Science.gov (United States)

    Dampney, R A L; Horiuchi, J

    2003-12-01

    Until about 10 years ago, knowledge of the functional organisation of the central pathways that subserve cardiovascular responses to homeostatic challenges and other stressors was based almost entirely on studies in anaesthetised animals. More recently, however, many studies have used the method of the expression of immediate early genes, particularly the c-fos gene, to identify populations of central neurons that are activated by such challenges in conscious animals. In this review we first consider the advantages and limitations of this method. Then, we discuss how the application of the method of immediate early gene expression, when used alone or in combination with other methods, has contributed to our understanding of the central mechanisms that regulate the autonomic and neuroendocrine response to various cardiovascular challenges (e.g., hypotension, hypoxia, hypovolemia, and other stressors) as they operate in the conscious state. In general, the results of studies of central cardiovascular pathways using immediate early gene expression are consistent with previous studies in anaesthetised animals, but in addition have revealed other previously unrecognised pathways that also contribute to cardiovascular regulation. Finally, we briefly consider recent evidence indicating that immediate early gene expression can modify the functional properties of central cardiovascular neurons, and the possible significance of this in producing long-term changes in the regulation of the cardiovascular system both in normal and pathological conditions.

  18. RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers

    Directory of Open Access Journals (Sweden)

    Van L.T. Hoang

    2017-08-01

    Full Text Available Identification of appropriate reference genes (RGs is critical to accurate data interpretation in quantitative real-time PCR (qPCR experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer.

  19. Studies of plant gene expression and function stimulated by space microgravity

    Science.gov (United States)

    Lu, Jinying; Liu, Min; Li, Huasheng; Zhao, Hui

    2016-07-01

    One of the important questions in space biology is how plants respond to an outer space environment i.e., how genetic expression is altered in space microgravity. In this study, the transcriptome of Arabidopsis thaliana seedlings was analyzed as part of the Germany SIMBOX (Science in Microgravity Box) spaceflight experiment on Shenzhou 8. A gene chip was used to screen gene expression differences in Arabidopsis thaliana seedlings between microgravity and 1g centrifugal force in space. Microarray analysis revealed that 368 genes were differentially expressed. Gene Ontology (GO) analysis indicated that these genes were involved in the plant's response to stress, secondary metabolism, hormone metabolism, transcription, protein phosphorylation, lipid metabolism, transport and cell wall metabolism processes. Real time PCR was used to analyzed the miRNA expression including Arabidopsis miR160,miR161, miR394, miR402, miR403, and miR408. MiR408 was significantly upregulated. An overexpression vector of Arabidopsis miR408 was constructed and transferred to Arabidopsis plant. The roots of plants over expressing miR408 exhibited a slower reorientation upon gravistimulation in comparison with those of wild-type. This result indicated that miR408 could play a role in root gravitropic response.

  20. Gene expression levels as endophenotypes in genome-wide association studies of Alzheimer disease

    Science.gov (United States)

    Zou, F.; Carrasquillo, M. M.; Pankratz, V. S.; Belbin, O.; Morgan, K.; Allen, M.; Wilcox, S. L.; Ma, L.; Walker, L. P.; Kouri, N.; Burgess, J. D.; Younkin, L. H.; Younkin, Samuel G.; Younkin, C. S.; Bisceglio, G. D.; Crook, J. E.; Dickson, D. W.; Petersen, R. C.; Graff-Radford, N.; Younkin, Steven G.; Ertekin-Taner, N.

    2010-01-01

    Background: Late-onset Alzheimer disease (LOAD) is a common disorder with a substantial genetic component. We postulate that many disease susceptibility variants act by altering gene expression levels. Methods: We measured messenger RNA (mRNA) expression levels of 12 LOAD candidate genes in the cerebella of 200 subjects with LOAD. Using the genotypes from our LOAD genome-wide association study for the cis-single nucleotide polymorphisms (SNPs) (n = 619) of these 12 LOAD candidate genes, we tested for associations with expression levels as endophenotypes. The strongest expression cis-SNP was tested for AD association in 7 independent case-control series (2,280 AD and 2,396 controls). Results: We identified 3 SNPs that associated significantly with IDE (insulin degrading enzyme) expression levels. A single copy of the minor allele for each significant SNP was associated with ∼twofold higher IDE expression levels. The most significant SNP, rs7910977, is 4.2 kb beyond the 3′ end of IDE. The association observed with this SNP was significant even at the genome-wide level (p = 2.7 × 10−8). Furthermore, the minor allele of rs7910977 associated significantly (p = 0.0046) with reduced LOAD risk (OR = 0.81 with a 95% CI of 0.70-0.94), as expected biologically from its association with elevated IDE expression. Conclusions: These results provide strong evidence that IDE is a late-onset Alzheimer disease (LOAD) gene with variants that modify risk of LOAD by influencing IDE expression. They also suggest that the use of expression levels as endophenotypes in genome-wide association studies may provide a powerful approach for the identification of disease susceptibility alleles. GLOSSARY AD = Alzheimer disease; CI = confidence interval; GWAS = genome-wide association study; LOAD = late-onset Alzheimer disease; mRNA = messenger RNA; OR = odds ratio; SNP = single nucleotide polymorphism. PMID:20142614

  1. Ascidian gene-expression profiles

    OpenAIRE

    Jeffery, William R.

    2002-01-01

    With the advent of gene-expression profiling, a large number of genes can now be investigated simultaneously during critical stages of development. This approach will be particularly informative in studies of ascidians, basal chordates whose genomes and embryology are uniquely suited for mapping developmental gene networks.

  2. Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR.

    Science.gov (United States)

    Obrero, Angeles; Die, Jose V; Román, Belén; Gómez, Pedro; Nadal, Salvador; González-Verdejo, Clara I

    2011-05-25

    The zucchini (Cucurbita pepo) is an important food crop, the transcriptomics of which are a fundamental tool to accelerate the development of new varieties by breeders. However, the suitability of reference genes for data normalization in zucchini has not yet been studied. The aim of this study was to assess the suitability of 13 genes for their potential use as reference genes in quantitative real-time PCR. Assays were performed on 34 cDNA samples representing plants under different stresses and at different developmental stages. The application of geNorm and NormFinder software revealed that the use of a combination of UFP, EF-1A, RPL36aA, PP2A, and CAC genes for the different experimental sets was the best strategy for reliable normalization. In contrast, 18S rRNA and TUA were less stable and unsuitable for use as internal controls. These results provide the possibility to allow more accurate use of qPCR in this horticultural crop.

  3. Comparative study of leptin and leptin receptor gene expression in different swine breeds.

    Science.gov (United States)

    Georgescu, S E; Manea, M A; Dinescu, S; Costache, M

    2014-02-14

    Leptin is an important regulator of appetite, energy metabolism, and reproduction and is mainly synthesized in the adipocytes and then secreted into the bloodstream. The leptin receptor was classified as type I cytokine receptor due to its structural homology with IL-6 receptors and the signaling pathways in which they are both involved. The aim of our study is to comparatively assess the gene expression levels of leptin (lep) and leptin receptor (lepr) in different swine breeds specialized either in meat production (Duroc, Belgian Landrace, Large White, Synthetic Lines LS-345, and LSP-2000) or fat production (Mangalitsa) in order to correlate them with morphological and productivity characteristics. Additionally, lepr pattern of expression was evaluated comparatively between different tissue types in the Mangalitsa breed. Our results revealed high expression of the lep gene in Mangalitsa compared to those of all the other breeds, while for the lepr gene, average/medium levels were registered in Mangalitsa and increased pattern of expression was found in the synthetic lines LS-345 and LSP-2000. Regarding the comparative analysis of lepr gene expression in various tissues in the Mangalitsa breed, elevated levels were found in the liver and kidney, while the lowest expression was identified in the brain and muscles. Our results suggest that the Mangalitsa population exhibits leptin resistance, which might be correlated with atypical morpho-productive characteristics for this breed, such as below-average prolificacy and a strong tendency to accumulate fat.

  4. Gene Expression in the Human Brain: The Current State of the Study of Specificity and Spatiotemporal Dynamics

    Science.gov (United States)

    Naumova, Oksana Yu.; Lee, Maria; Rychkov, Sergei Yu.; Vlasova, Natalia V.; Grigorenko, Elena L.

    2013-01-01

    Gene expression is one of the main molecular processes regulating the differentiation, development, and functioning of cells and tissues. In this review a handful of relevant terms and concepts are introduced and the most common techniques used in studies of gene expression/expression profiling (also referred to as studies of the transcriptome or…

  5. Gene Expression in the Human Brain: The Current State of the Study of Specificity and Spatiotemporal Dynamics

    Science.gov (United States)

    Naumova, Oksana Yu.; Lee, Maria; Rychkov, Sergei Yu.; Vlasova, Natalia V.; Grigorenko, Elena L.

    2013-01-01

    Gene expression is one of the main molecular processes regulating the differentiation, development, and functioning of cells and tissues. In this review a handful of relevant terms and concepts are introduced and the most common techniques used in studies of gene expression/expression profiling (also referred to as studies of the transcriptome or…

  6. Study on the protein expression and amplification of HER2 gene in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Sunan Wang; Yingying Li; Zhengshun Xu; Wenzhao Zhao; Tian Yun; Wuling Zhu; Yangkun Wang

    2014-01-01

    Objective:The aim of the study was to investigate the human epidermal growth factor receptor 2 (HER2) gene amplification and protein expression and interpretation points in the stomach mixed carcinomas. Methods:Immunohisto-chemistry (IHC) and fluorescence in situ hybridization (FISH) technique were used to detect HER2 gene amplification and ex-pression of HER2 protein in 442 cases of gastric mixed carcinoma. Results:The expression rate of HER2 protein was 41.2%(182/442):the HER2 protein expression IHC 3+extensive type in 18 cases, partial type in 21 cases, focal type in 8 cases, accounting for 10.6%(47/442);the HER2 protein expression IHC 2+extensive type in 23 cases, partial type in 28 cases, focal type in 11 cases, accounting for 14.0%(62/442);the HER2 protein expression IHC 1+extensive type in 27 cases, partial type in 31 cases, focal type in 15 cases, accounting for 16.5%(73/442). HER2 gene amplification rate of 442 cases was 16.1%(71/442). In 182 cases of HER2 protein positive expression, the HER2 gene cluster amplification rate was 14.8%(27/182), large granular amplification rate 11.0%(20/182), punctate amplification rate 6.0%(11/182) and high polysomy 7.1%(13/182). In 71 cases of HER2 gene amplification, there was 42 cases of HER2 protein expression IHC 3+, 22 cases of HER2 protein expression IHC 2+, and 7 cases of IHC 1+. Conclusion:HER2 detection of gastric mixed carcinoma has great heterogeneity, HER2 protein positive expression is divided into extensive type, partial type and focal type, and HER2 gene positive amplifica-tion is divided into cluster amplification, large granular amplification, punctate amplification and high polysomy. These typing of HER2 protein expression and HER2 gene amplification provide reference index to quantify for targeted therapeutic ef ect of anticancer drugs.

  7. Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

    Science.gov (United States)

    Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

    2015-06-01

    A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages.

  8. A Model System for the Study of Gene Expression in the Undergraduate Laboratory

    Science.gov (United States)

    Hargadon, Kristian M.

    2016-01-01

    The flow of genetic information from DNA to RNA to protein, otherwise known as the "central dogma" of biology, is one of the most basic and overarching concepts in the biological sciences. Nevertheless, numerous studies have reported student misconceptions at the undergraduate level of this fundamental process of gene expression. This…

  9. Maternal obesity affects fetal neurodevelopmental and metabolic gene expression: a pilot study.

    Directory of Open Access Journals (Sweden)

    Andrea G Edlow

    Full Text Available OBJECTIVE: One in three pregnant women in the United States is obese. Their offspring are at increased risk for neurodevelopmental and metabolic morbidity. Underlying molecular mechanisms are poorly understood. We performed a global gene expression analysis of mid-trimester amniotic fluid cell-free fetal RNA in obese versus lean pregnant women. METHODS: This prospective pilot study included eight obese (BMI≥30 and eight lean (BMI<25 women undergoing clinically indicated mid-trimester genetic amniocentesis. Subjects were matched for gestational age and fetal sex. Fetuses with abnormal karyotype or structural anomalies were excluded. Cell-free fetal RNA was extracted from amniotic fluid and hybridized to whole genome expression arrays. Genes significantly differentially regulated in 8/8 obese-lean pairs were identified using paired t-tests with the Benjamini-Hochberg correction (false discovery rate of <0.05. Biological interpretation was performed with Ingenuity Pathway Analysis and the BioGPS gene expression atlas. RESULTS: In fetuses of obese pregnant women, 205 genes were significantly differentially regulated. Apolipoprotein D, a gene highly expressed in the central nervous system and integral to lipid regulation, was the most up-regulated gene (9-fold. Apoptotic cell death was significantly down-regulated, particularly within nervous system pathways involving the cerebral cortex. Activation of the transcriptional regulators estrogen receptor, FOS, and STAT3 was predicted in fetuses of obese women, suggesting a pro-estrogenic, pro-inflammatory milieu. CONCLUSION: Maternal obesity affects fetal neurodevelopmental and metabolic gene expression as early as the second trimester. These findings may have implications for postnatal neurodevelopmental and metabolic abnormalities described in the offspring of obese women.

  10. Docetaxel-Chitosan nanoparticles for breast cancer treatment: cell viability and gene expression study.

    Science.gov (United States)

    Mirzaie, Zahra H; Irani, Shiva; Mirfakhraie, Reza; Atyabi, Seyed Mohammad; Dinarvand, Meshkat; Dinarvand, Rassoul; Varshochian, Reyhaneh; Atyabi, Fatemeh

    2016-12-01

    Docetaxel acts through the inhibition of tubulin polymerization and reduction in the expression of BCL-2 gene. In this study, nanoparticles containing Docetaxel were prepared and their effects on the gene expression levels of BCL-2 and BAX genes were investigated. The drug was first conjugated to chitosan, and the nanoparticles were assembled in the presence of hyaluronic acid. Conjugations were confirmed by (1) H-NMR, and the obtained nanoparticles were characterized by dynamic light scattering and SEM. Cytotoxicity of the nanoparticles, cellular uptake, and cell death were evaluated. Finally, the effect of nanoparticles on the expression of BAX and BCL-2 genes in MCF-7 cells were investigated through real-time PCR. The results revealed that the prepared NPs had spherical shape with narrow size distribution of nanoparticles and free Docetaxel investigations revealed that increasing the treatment time with nanoparticles led to decrease in the rate of cell viability. BAX and BCL-2 gene expressions were decreased in nanoparticle-treated cells in comparison with intact cells, while the BAX/BCL-2 ratio was significantly elevated compared with free drug-treated cells after 72 h. Docetaxel-conjugated NPs may offer a promising treatment with low off-target toxicity for breast cancer. © 2016 John Wiley & Sons A/S.

  11. A phase I study of hydralazine to demethylate and reactivate the expression of tumor suppressor genes

    Directory of Open Access Journals (Sweden)

    Trejo-Becerril Catalina

    2005-04-01

    Full Text Available Abstract Background The antihypertensive compound hydralazine is a known demethylating agent. This phase I study evaluated the tolerability and its effects upon DNA methylation and gene reactivation in patients with untreated cervical cancer. Methods Hydralazine was administered to cohorts of 4 patients at the following dose levels: I 50 mg/day, II 75 mg/day, III 100 mg/day and IV 150 mg/day. Tumor biopsies and peripheral blood samples were taken the day before and after treatment. The genes APC, MGMT; ER, GSTP1, DAPK, RARβ, FHIT and p16 were evaluated pre and post-treatment for DNA promoter methylation and gene expression by MSP (Methylation-Specific PCR and RT-PCR respectively in each of the tumor samples. Methylation of the imprinted H19 gene and the "normally methylated" sequence clone 1.2 was also analyzed. Global DNA methylation was analyzed by capillary electrophoresis and cytosine extension assay. Toxicity was evaluated using the NCI Common Toxicity Criteria. Results Hydralazine was well tolerated. Toxicities were mild being the most common nausea, dizziness, fatigue, headache and palpitations. Overall, 70% of the pretreatment samples and all the patients had at least one methylated gene. Rates of demethylation at the different dose levels were as follows: 50 mg/day, 40%; 75 mg/day, 52%, 100 mg/day, 43%, and 150 mg/day, 32%. Gene expression analysis showed only 12 informative cases, of these 9 (75% re-expressed the gene. There was neither change in the methylation status of H19 and clone 1.2 nor changes in global DNA methylation. Conclusion Hydralazine at doses between 50 and 150 mg/day is well tolerated and effective to demethylate and reactivate the expression of tumor suppressor genes without affecting global DNA methylation

  12. Oligomicroarray-based primary study of gene expression profile changes in Barrett's esophagus

    Institute of Scientific and Technical Information of China (English)

    Wang Xingwei; Sun Yonggang; Xu Mei; Fang Dianchun; Gao Hengjun; Xu Jiangtao

    2008-01-01

    Objective: To analyze the differential expression genes (DEGs) between Barrett's esophagus (BE) and normal esophagus mucosa and explore the target genes related to the development and progression of BE. Methods: The total RNAs of matched BE and normal esophagus mucosa of BE patients were isolated using one step Trizol method.Matched RNAs were qualified using 10 g/L agarose gel electrophoresis, cRNAs were synthesized, fluorescence labeled and purified after total RNAs were purified. The RNAs of BE and normal esophagus mucosa were hybridized with Agilent oligomicroarray (30 968 probes). The fluorescence intensity features were detected by Agilent scanner and quantified by feature extraction software. Results: (1) The total RNA, reverse transcription product and fluorescence labeled cRNA were all of high quality; (2) There were 142 up-regulated genes and 284 down-regulated genes among 2-fold DEGs. Conclusion: Microarray-based studies are feasible in endoscopically obtained tissues. Many BE-associated genes are screened by the high-throughput gene chip. The development and progression of BE is a complicated process involving multiple genes and multiple procedures, and functional study of these genes may help to identify the key genes or pathways involved in the pathogenesis and development of BE.

  13. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  14. Gene Expression Analysis of In Vitro Cocultures to Study Interactions between Breast Epithelium and Stroma

    Directory of Open Access Journals (Sweden)

    Patricia Casbas-Hernandez

    2011-01-01

    Full Text Available The interactions between breast epithelium and stroma are fundamental to normal tissue homeostasis and for tumor initiation and progression. Gene expression studies of in vitro coculture models demonstrate that in vitro models have relevance for tumor progression in vivo. For example, stromal gene expression has been shown to vary in association with tumor subtype in vivo, and analogous in vitro cocultures recapitulate subtype-specific biological interactions. Cocultures can be used to study cancer cell interactions with specific stromal components (e.g., immune cells, fibroblasts, endothelium and different representative cell lines (e.g., cancer-associated versus normal-associated fibroblasts versus established, immortalized fibroblasts can help elucidate the role of stromal variation in tumor phenotypes. Gene expression data can also be combined with cell-based assays to identify cellular phenotypes associated with gene expression changes. Coculture systems are manipulable systems that can yield important insights about cell-cell interactions and the cellular phenotypes that occur as tumor and stroma co-evolve.

  15. Tumour sampling method can significantly influence gene expression profiles derived from neoadjuvant window studies.

    Science.gov (United States)

    Pearce, Dominic A; Arthur, Laura M; Turnbull, Arran K; Renshaw, Lorna; Sabine, Vicky S; Thomas, Jeremy S; Bartlett, John M S; Dixon, J Michael; Sims, Andrew H

    2016-07-07

    Patient-matched transcriptomic studies using tumour samples before and after treatment allow inter-patient heterogeneity to be controlled, but tend not to include an untreated comparison. Here, Illumina BeadArray technology was used to measure dynamic changes in gene expression from thirty-seven paired diagnostic core and surgically excised breast cancer biopsies obtained from women receiving no treatment prior to surgery, to determine the impact of sampling method and tumour heterogeneity. Despite a lack of treatment and perhaps surprisingly, consistent changes in gene expression were identified during the diagnosis-surgery interval (48 up, 2 down; Siggenes FDR 0.05) in a manner independent of both subtype and sampling-interval length. Instead, tumour sampling method was seen to directly impact gene expression, with similar effects additionally identified in six published breast cancer datasets. In contrast with previous findings, our data does not support the concept of a significant wounding or immune response following biopsy in the absence of treatment and instead implicates a hypoxic response following the surgical biopsy. Whilst sampling-related gene expression changes are evident in treated samples, they are secondary to those associated with response to treatment. Nonetheless, sampling method remains a potential confounding factor for neoadjuvant study design.

  16. A Comparative Study of Feature Selection and Classification Methods for Gene Expression Data of Glioma

    KAUST Repository

    Abusamra, Heba

    2013-11-01

    Microarray gene expression data gained great importance in recent years due to its role in disease diagnoses and prognoses which help to choose the appropriate treatment plan for patients. This technology has shifted a new era in molecular classification. Interpreting gene expression data remains a difficult problem and an active research area due to their native nature of “high dimensional low sample size”. Such problems pose great challenges to existing classification methods. Thus, effective feature selection techniques are often needed in this case to aid to correctly classify different tumor types and consequently lead to a better understanding of genetic signatures as well as improve treatment strategies. This paper aims on a comparative study of state-of-the- art feature selection methods, classification methods, and the combination of them, based on gene expression data. We compared the efficiency of three different classification methods including: support vector machines, k-nearest neighbor and random forest, and eight different feature selection methods, including: information gain, twoing rule, sum minority, max minority, gini index, sum of variances, t-statistics, and one-dimension support vector machine. Five-fold cross validation was used to evaluate the classification performance. Two publicly available gene expression data sets of glioma were used in the experiments. Results revealed the important role of feature selection in classifying gene expression data. By performing feature selection, the classification accuracy can be significantly boosted by using a small number of genes. The relationship of features selected in different feature selection methods is investigated and the most frequent features selected in each fold among all methods for both datasets are evaluated.

  17. Profiling gene expression in human placentae of different gestational ages: an OPRU Network and UW SCOR Study.

    Science.gov (United States)

    Mikheev, Andrei M; Nabekura, Tomohiro; Kaddoumi, Amal; Bammler, Theo K; Govindarajan, Rajgopal; Hebert, Mary F; Unadkat, Jashvant D

    2008-11-01

    We used the whole-genome approach to identify major functional categories of genes whose expression depends on gestational age. Using microarray analysis, we compared gene expression profiles in the villous tissues of first (45-59 days) and second trimester (109-115 days) placentae with C-section term placentae. We found that in first trimester placentae, genes related to cell cycle, DNA, amino acids, and carbohydrate metabolism were significantly overrepresented, while genes related to signal transduction were underrepresented. Among genes involved in organism defense, we identified genes involved in chemical response, metabolism, and transport. Analysis of signal transduction pathways suggested, and subsequently confirmed independently, that the Wnt pathway was changed with gestational age leading to inhibition of beta-catenin protein expression. Our study will serve as a reference database to gain insight into the regulation of gene expression in the developing placentae and to compare with gene expression in placentae from complicated pregnancies.

  18. Identification of suitable reference genes for gene expression studies using quantitative polymerase chain reaction in lung cancer in vitro.

    Science.gov (United States)

    Ali, Hassan; Du, Zhenwu; Li, Xiuying; Yang, Qiwei; Zhang, Yu Cheng; Wu, Mei; Li, Yi; Zhang, Guizhen

    2015-05-01

    The present study aimed to examine 10 housekeeping genes (HKGs), including 18s ribosomal RNA (18S), glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH), ribosomal protein large P0 (RPLP0), β‑actin (ACTB), peptidylprolyl isomerase A (PPIA), phosphoglycerate kinase‑1 (PGK1), β‑2‑microglobulin (B2M), ribosomal protein LI3a (RPL13A), hypoxanthine phosphoribosyl transferase‑1 (HPRT1) and TATA box binding protein (TBP) in order to identify the most stable and suitable reference genes for use in expression studies in non‑small cell lung cancer. The mRNA expression encoding the panel of the 10 HKGs was determined using reverse transcription‑quantitative PCR (RT‑qPCR) in human lung cancer cell lines. Three software programs, BestKeeper, NormFinder and geNorm, were used to ascertain the most suitable reference genes to normalize the RNA input. The present study examined three lung cancer cell lines (A549, NCI‑H446 and NCI‑H460). The analysis of the experimental data using BestKeeper software revealed that all 10 HKGs were stable, with GADPH, followed by 18S being the most stable genes and PPIA and HPRT1 being the least stable genes. The NormFinder software results demonstrated that PPIA followed by ACTB were the most stable and B2M and RPLP0 were the least stable. The geNorm software results revealed that ACTB and PGK1, followed by PPIA were the most stable genes and B2M and RPLP0 were identified as the least stable genes. Due to discrepancies in the ranking orders of the reference genes obtained by different analyzing software programs, it was not possible to determine a single universal reference gene. The suitability of selected reference genes requires unconditional validation prior to each study. Based on the three analyzing programs, ACTB, PPIA and PGK1 were the most stable reference genes in lung cancer cell lines.

  19. SVAw - a web-based application tool for automated surrogate variable analysis of gene expression studies.

    Science.gov (United States)

    Pirooznia, Mehdi; Seifuddin, Fayaz; Goes, Fernando S; Leek, Jeffrey T; Zandi, Peter P

    2013-03-11

    Surrogate variable analysis (SVA) is a powerful method to identify, estimate, and utilize the components of gene expression heterogeneity due to unknown and/or unmeasured technical, genetic, environmental, or demographic factors. These sources of heterogeneity are common in gene expression studies, and failing to incorporate them into the analysis can obscure results. Using SVA increases the biological accuracy and reproducibility of gene expression studies by identifying these sources of heterogeneity and correctly accounting for them in the analysis. Here we have developed a web application called SVAw (Surrogate variable analysis Web app) that provides a user friendly interface for SVA analyses of genome-wide expression studies. The software has been developed based on open source bioconductor SVA package. In our software, we have extended the SVA program functionality in three aspects: (i) the SVAw performs a fully automated and user friendly analysis workflow; (ii) It calculates probe/gene Statistics for both pre and post SVA analysis and provides a table of results for the regression of gene expression on the primary variable of interest before and after correcting for surrogate variables; and (iii) it generates a comprehensive report file, including graphical comparison of the outcome for the user. SVAw is a web server freely accessible solution for the surrogate variant analysis of high-throughput datasets and facilitates removing all unwanted and unknown sources of variation. It is freely available for use at http://psychiatry.igm.jhmi.edu/sva. The executable packages for both web and standalone application and the instruction for installation can be downloaded from our web site.

  20. Expression studies of six human obesity-related genes in seven tissues from divergent pig breeds.

    Science.gov (United States)

    Cirera, S; Jensen, M S; Elbrønd, V S; Moesgaard, S G; Christoffersen, B Ø; Kadarmideen, H N; Skovgaard, K; Bruun, C V; Karlskov-Mortensen, P; Jørgensen, C B; Fredholm, M

    2014-02-01

    Obesity has reached epidemic proportions globally and has become the cause of several major health risks worldwide. Presently, more than 100 loci have been related to obesity and metabolic traits in humans by genome-wide association studies. The complex genetic architecture behind obesity has triggered a need for the development of better animal models than rodents. The pig has emerged as a very promising biomedical model to study human obesity traits. In this study, we have characterized the expression patterns of six obesity-related genes, leptin (LEP), leptin receptor (LEPR), melanocortin 4 receptor (MC4R), fat mass and obesity associated (FTO), neuronal growth regulator 1 (NEGR)1 and adiponectin (ADIPOQ), in seven obesity-relevant tissues (liver; muscle; pancreas; hypothalamus; and retroperitoneal, subcutaneous and mesenteric adipose tissues) in two pig breeds (production pigs and Göttingen minipigs) that deviate phenotypically and genetically from each other with respect to obesity traits. We observe significant differential expression for LEP, LEPR and ADIPOQ in muscle and in all three adipose tissues. Interestingly, in pancreas, LEP expression is only detected in the fat minipigs. FTO shows significant differential expression in all tissues analyzed, and NEGR1 shows significant differential expression in muscle, pancreas, hypothalamus and subcutaneous adipose tissue. The MC4R transcript can be detected only in hypothalamus. In general, the expression profiles of the investigated genes are in accordance with those observed in human studies. Our study shows that both the differences between the investigated breeds and the phenotypic state with respect to obesity/leanness play a large role for differential expression of the obesity-related genes.

  1. Selection and validation of reference genes for miRNA expression studies during porcine pregnancy.

    Directory of Open Access Journals (Sweden)

    Jocelyn M Wessels

    Full Text Available MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A

  2. Genes and gene expression: Localization, damage and control: A multilevel and inter-disciplinary study

    Energy Technology Data Exchange (ETDEWEB)

    Ts' o, P.O.P.

    1990-09-01

    The main objectives of this Program Project is to develop strategy and technology for the study of gene structure, organization and function in a multi-disciplinary, highly coordinated manner. In Project I, Molecular Cytology, the establishment of all instrumentation for the computerized microscopic imaging system (CMIS) has been completed with the software in place, including measurement of the third dimension (along the Z-axis). The technique is now at hand to measure single copy DNA in the nucleus, single copy mRNA in the cell, and finally, we are in the process of developing mathematical approaches for the analysis of the relative spatial 3-D relationship among the chromosomes and the individual genes in the interphasal nucleus. Also, we have a sensitive and reliable method for measuring single-stranded DNA breaks which will be useful for the determination of damage to DNA caused by ionizing radiation. In Project II, the mapping of restriction fragments by 2-D enzymatic and electrophoretic analysis has been perfected for application. In Project III, a major finding is that the binding constant and effectiveness of antisense oligonucleotide analogues, Matagen, can be significantly improved by substituting 2{prime}-O-methylribos methylphosphonate backbones for the current 2{prime}-deoxyribomethylphosphonate backbones. 15 refs., 10 figs., 2 tabs.

  3. Study on the relationship between the RAR-β gene expressive defection and its methylation

    Institute of Scientific and Technical Information of China (English)

    高艳萍; 李敏; 张颖颖; 王翰; 贺小红; 王泽华

    2007-01-01

    Objective To observe the expression of RAR-β gene in SiHa, HeLa,C33A and CasKi cell lines of cervical carcinoma and to investigate the role of methylated RAR-β in its expressive defection. Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of RAR-β gene. Immunohistochemistry and Western Blot were used to analyze the protein expression of RAR-β gene in four cervical cancer cell lines as well as the influence of 5-Aza-cdR on gene expressive defection. Meth...

  4. Reference gene selection for quantitative gene expression studies during biological invasions: A test on multiple genes and tissues in a model ascidian Ciona savignyi.

    Science.gov (United States)

    Huang, Xuena; Gao, Yangchun; Jiang, Bei; Zhou, Zunchun; Zhan, Aibin

    2016-01-15

    As invasive species have successfully colonized a wide range of dramatically different local environments, they offer a good opportunity to study interactions between species and rapidly changing environments. Gene expression represents one of the primary and crucial mechanisms for rapid adaptation to local environments. Here, we aim to select reference genes for quantitative gene expression analysis based on quantitative Real-Time PCR (qRT-PCR) for a model invasive ascidian, Ciona savignyi. We analyzed the stability of ten candidate reference genes in three tissues (siphon, pharynx and intestine) under two key environmental stresses (temperature and salinity) in the marine realm based on three programs (geNorm, NormFinder and delta Ct method). Our results demonstrated only minor difference for stability rankings among the three methods. The use of different single reference gene might influence the data interpretation, while multiple reference genes could minimize possible errors. Therefore, reference gene combinations were recommended for different tissues - the optimal reference gene combination for siphon was RPS15 and RPL17 under temperature stress, and RPL17, UBQ and TubA under salinity treatment; for pharynx, TubB, TubA and RPL17 were the most stable genes under temperature stress, while TubB, TubA and UBQ were the best under salinity stress; for intestine, UBQ, RPS15 and RPL17 were the most reliable reference genes under both treatments. Our results suggest that the necessity of selection and test of reference genes for different tissues under varying environmental stresses. The results obtained here are expected to reveal mechanisms of gene expression-mediated invasion success using C. savignyi as a model species.

  5. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene ...

  6. Bayesian coclustering of Anopheles gene expression time series: Study of immune defense response to multiple experimental challenges

    OpenAIRE

    Heard, Nicholas A.; Holmes, Christopher C.; Stephens, David A.; Hand, David J.; Dimopoulos, George

    2005-01-01

    We present a method for Bayesian model-based hierarchical coclustering of gene expression data and use it to study the temporal transcription responses of an Anopheles gambiae cell line upon challenge with multiple microbial elicitors. The method fits statistical regression models to the gene expression time series for each experiment and performs coclustering on the genes by optimizing a joint probability model, characterizing gene coregulation between multiple experiments. We compute the mo...

  7. Human Lacrimal Gland Gene Expression

    Science.gov (United States)

    Aakalu, Vinay Kumar; Parameswaran, Sowmya; Maienschein-Cline, Mark; Bahroos, Neil; Shah, Dhara; Ali, Marwan; Krishnakumar, Subramanian

    2017-01-01

    Background The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development. Methods We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium. Results The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described. Conclusions Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas. PMID:28081151

  8. Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

    Science.gov (United States)

    Marcial-Quino, Jaime; Fierro, Francisco; De la Mora-De la Mora, Ignacio; Enríquez-Flores, Sergio; Gómez-Manzo, Saúl; Vanoye-Carlo, America; Garcia-Torres, Itzhel; Sierra-Palacios, Edgar; Reyes-Vivas, Horacio

    2016-04-25

    The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia.

  9. A replication study for genome-wide gene expression levels in two layer lines elucidates differentially expressed genes of pathways involved in bone remodeling and immune responsiveness.

    Directory of Open Access Journals (Sweden)

    Christin Habig

    Full Text Available The current replication study confirmed significant differences in gene expression profiles of the cerebrum among the two commercial layer lines Lohmann Selected Leghorn (LSL and Lohmann Brown (LB. Microarray analyses were performed for 30 LSL and another 30 LB laying hens kept in the small group housing system Eurovent German. A total of 14,103 microarray probe sets using customized Affymetrix ChiGene-1_0-st Arrays with 20,399 probe sets were differentially expressed among the two layer lines LSL and LB (FDR adjusted P-value <0.05. An at least 2-fold change in expression levels could be observed for 388 of these probe sets. In LSL, 214 of the 388 probe sets were down- and 174 were up-regulated and vice versa for the LB layer line. Among the 174 up-regulated probe sets in LSL, we identified 51 significantly enriched Gene ontology (GO terms of the biological process category. A total of 63 enriched GO-terms could be identified for the 214 down-regulated probe sets of the layer line LSL. We identified nine genes significantly differentially expressed between the two layer lines in both microarray experiments. These genes play a crucial role in protection of neuronal cells from oxidative stress, bone mineral density and immune response among the two layer lines LSL and LB. Thus, the different regulation of these genes may significantly contribute to phenotypic trait differences among these layer lines. In conclusion, these novel findings provide a basis for further research to improve animal welfare in laying hens and these layer lines may be of general interest as an animal model.

  10. Transcriptome sequencing study implicates immune-related genes differentially expressed in schizophrenia: new data and a meta-analysis

    Science.gov (United States)

    Sanders, A R; Drigalenko, E I; Duan, J; Moy, W; Freda, J; Göring, H H H; Gejman, P V

    2017-01-01

    We undertook an RNA sequencing (RNAseq)-based transcriptomic profiling study on lymphoblastoid cell lines of a European ancestry sample of 529 schizophrenia cases and 660 controls, and found 1058 genes to be differentially expressed by affection status. These differentially expressed genes were enriched for involvement in immunity, especially the 697 genes with higher expression in cases. Comparing the current RNAseq transcriptomic profiling to our previous findings in an array-based study of 268 schizophrenia cases and 446 controls showed a highly significant positive correlation over all genes. Fifteen (18%) of the 84 genes with significant (false discovery rateRNAseq and array data sets (797 cases and 1106 controls) showed 169 additional genes (besides those found in the primary RNAseq-based analysis) to be differentially expressed, and provided further evidence of immune gene enrichment. In addition to strengthening our previous array-based gene expression differences in schizophrenia cases versus controls and providing transcriptomic support for some genes implicated by other approaches for schizophrenia, our study detected new genes differentially expressed in schizophrenia. We highlight RNAseq-based differential expression of various genes involved in neurodevelopment and/or neuronal function, and discuss caveats of the approach. PMID:28418402

  11. A contribution to the study of plant development evolution based on gene co-expression networks

    Directory of Open Access Journals (Sweden)

    Francisco J. Romero-Campero

    2013-08-01

    Full Text Available Phototrophic eukaryotes are among the most successful organisms on Earth due to their unparalleled efficiency at capturing light energy and fixing carbon dioxide to produce organic molecules. A conserved and efficient network of light-dependent regulatory modules could be at the bases of this success. This regulatory system conferred early advantages to phototrophic eukaryotes that allowed for specialization, complex developmental processes and modern plant characteristics. We have studied light-dependent gene regulatory modules from algae to plants employing integrative-omics approaches based on gene co-expression networks. Our study reveals some remarkably conserved ways in which eukaryotic phototrophs deal with day length and light signaling. Here we describe how a family of Arabidopsis transcription factors involved in photoperiod response has evolved from a single algal gene according to the innovation, amplification and divergence theory of gene evolution by duplication. These modifications of the gene co-expression networks from the ancient unicellular green algae Chlamydomonas reinhardtii to the modern brassica Arabidopsis thaliana may hint on the evolution and specialization of plants and other organisms.

  12. Identification of reference genes for gene expression studies during seed germination and seedling establishment Ricinus communis L.

    NARCIS (Netherlands)

    Ribeiro de Jesus, P.R.; Dekkers, S.J.W.; Fernandez, L.G.; Castro, De R.D.; Ligterink, W.; Hilhorst, H.W.M.

    2014-01-01

    Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of re

  13. Identification of reference genes for gene expression studies during seed germination and seedling establishment Ricinus communis L.

    NARCIS (Netherlands)

    Ribeiro de Jesus, P.R.; Dekkers, S.J.W.; Fernandez, L.G.; Castro, De R.D.; Ligterink, W.; Hilhorst, H.W.M.

    2014-01-01

    Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of

  14. Expression and replication studies to identify new candidate genes involved in normal hearing function.

    Science.gov (United States)

    Girotto, Giorgia; Vuckovic, Dragana; Buniello, Annalisa; Lorente-Cánovas, Beatriz; Lewis, Morag; Gasparini, Paolo; Steel, Karen P

    2014-01-01

    Considerable progress has been made in identifying deafness genes, but still little is known about the genetic basis of normal variation in hearing function. We recently carried out a Genome Wide Association Study (GWAS) of quantitative hearing traits in southern European populations and found several SNPs with suggestive but none with significant association. In the current study, we followed up these SNPs to investigate which of them might show a genuine association with auditory function using alternative approaches. Firstly, we generated a shortlist of 19 genes from the published GWAS results. Secondly, we carried out immunocytochemistry to examine expression of these 19 genes in the mouse inner ear. Twelve of them showed distinctive cochlear expression patterns. Four showed expression restricted to sensory hair cells (Csmd1, Arsg, Slc16a6 and Gabrg3), one only in marginal cells of the stria vascularis (Dclk1) while the others (Ptprd, Grm8, GlyBP, Evi5, Rimbp2, Ank2, Cdh13) in multiple cochlear cell types. In the third step, we tested these 12 genes for replication of association in an independent set of samples from the Caucasus and Central Asia. Nine out of them showed nominally significant association (pgene-based test. Finally, to look for genotype-phenotype relationship, the audiometric profiles of the three genotypes of the most strongly associated gene variants were analyzed. Seven out of the 9 replicated genes (CDH13, GRM8, ANK2, SLC16A6, ARSG, RIMBP2 and DCLK1) showed an audiometric pattern with differences between different genotypes further supporting their role in hearing function. These data demonstrate the usefulness of this multistep approach in providing new insights into the molecular basis of hearing and may suggest new targets for treatment and prevention of hearing impairment.

  15. Gene expression profiling of MPP+-treated MN9D cells: a mechanism of toxicity study.

    Science.gov (United States)

    Wang, Jianyong; Xu, Zengjun; Fang, Hong; Duhart, Helen M; Patterson, Tucker A; Ali, Syed F

    2007-09-01

    Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of midbrain dopaminergic neurons with unknown etiology. MPP+ (1-methyl-4-phenylpyridinium) is the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like syndromes in humans and animals. MPTP/MPP+ treatment produces selective dopaminergic neuronal degeneration, therefore, these agents are commonly used to study the pathogenesis of PD. However, the mechanisms of their toxicity have not been elucidated. In order to gain insights into MPP+-induced neurotoxicity, a gene expression microarray study was performed using a midbrain-derived dopaminergic neuronal cell line, MN9D. Utilizing a two-color reference design, Agilent mouse oligonucleotide microarrays were used to examine relative gene expression changes in MN9D cells treated with 40microM MPP+ compared with controls. Bioinformatics tools were used for data evaluation. Briefly, raw data were imported into the NCTR ArrayTrack database, normalized using a Lowess method and data quality was assessed. The Student's t-test was used to determine significant changes in gene expression (set as p1.5). Gene Ontology for Function Analysis (GOFFA) and Ingenuity Pathway Analysis were employed to analyze the functions and roles of significant genes in biological processes. Of the 51 significant genes identified, 44 were present in the GOFFA or Ingenuity database. These data indicate that multiple pathways are involved in the underlying mechanisms of MPP+-induced neurotoxicity, including apoptosis, oxidative stress, iron binding, cellular metabolism, and signal transduction. These data also indicate that MPP+-induced toxicity shares common molecular mechanisms with the pathogenesis of PD and further pathway analyses will be conducted to explore these mechanisms.

  16. Advances in plant cell type-specific genome-wide studies of gene expression

    Institute of Scientific and Technical Information of China (English)

    Ying WANG; Yuling JIAO

    2011-01-01

    Cell is the functional unit of life.To study the complex interactions of systems of biological molecules,it is crucial to dissect these molecules at the cell level.In recent years,major progresses have been made by plant biologists to profile gene expression in specific cell types at the genome-wide level.Approaches based on the isolation of cells,polysomes or nuclei have been developed and successfully used for studying the cell types from distinct organs of several plant species.These cell-level data sets revealed previously unrecognized cellular properties,such as cell-specific gene expression modules and hormone response centers,and should serve as essential resources for functional genomic analyses.Newly developed technologies are more affordable to many laboratories and should help to provide new insights at the cellular resolution in the near future.

  17. Sex hormones and gene expression signatures in peripheral blood from postmenopausal women - the NOWAC postgenome study

    Directory of Open Access Journals (Sweden)

    Rylander Charlotta

    2011-03-01

    Full Text Available Abstract Background Postmenopausal hormone therapy (HT influences endogenous hormone concentrations and increases the risk of breast cancer. Gene expression profiling may reveal the mechanisms behind this relationship. Our objective was to explore potential associations between sex hormones and gene expression in whole blood from a population-based, random sample of postmenopausal women Methods Gene expression, as measured by the Applied Biosystems microarray platform, was compared between hormone therapy (HT users and non-users and between high and low hormone plasma concentrations using both gene-wise analysis and gene set analysis. Gene sets found to be associated with HT use were further analysed for enrichment in functional clusters and network predictions. The gene expression matrix included 285 samples and 16185 probes and was adjusted for significant technical variables. Results Gene-wise analysis revealed several genes significantly associated with different types of HT use. The functional cluster analyses provided limited information on these genes. Gene set analysis revealed 22 gene sets that were enriched between high and low estradiol concentration (HT-users excluded. Among these were seven oestrogen related gene sets, including our gene list associated with systemic estradiol use, which thereby represents a novel oestrogen signature. Seven gene sets were related to immune response. Among the 15 gene sets enriched for progesterone, 11 overlapped with estradiol. No significant gene expression patterns were found for testosterone, follicle stimulating hormone (FSH or sex hormone binding globulin (SHBG. Conclusions Distinct gene expression patterns associated with sex hormones are detectable in a random group of postmenopausal women, as demonstrated by the finding of a novel oestrogen signature.

  18. Selection of reliable reference genes for gene expression studies in Clonostachys rosea 67-1 under sclerotial induction.

    Science.gov (United States)

    Sun, Zhan-Bin; Li, Shi-Dong; Sun, Man-Hong

    2015-07-01

    Reference genes are important to precisely quantify gene expression by real-time PCR. In order to identify stable and reliable expressed genes in mycoparasite Clonostachys rosea in different modes of nutrition, seven commonly used housekeeping genes, 18S rRNA, actin, β-tubulin, elongation factor 1, ubiquitin, ubiquitin-conjugating enzyme and glyceraldehyde-3-phosphate dehydrogenase, from the effective biocontrol isolate C. rosea 67-1 were tested for their expression under sclerotial induction and during vegetative growth on PDA medium. Analysis by three software programs showed that differences existed among the candidates. Elongation factor 1 was most stable; the M value in geNorm, SD value in Bestkeeper and stability value in Normfinder analysis were 0.405, 0.450 and 0.442, respectively, indicating that the gene elongation factor 1 could be used to normalize gene expression in C. rosea in both vegetative growth and parasitic process. By using elongation factor 1, the expression of a serine protease gene, sep, in different conditions was assessed, which was consistent with the transcriptomic data. This research provides an effective method to quantitate expression changes of target genes in C. rosea, and will assist in further investigation of parasitism-related genes of this fungus.

  19. Study on the polymorphisms and promoter methylation and expression of the glutathione Stransferases P1 gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    张友才

    2006-01-01

    Objective To study the relationship between hepatocellular carcinoma (HCC) and the polymorphisms, promoter methylation, and expression of glutathione S-transferases P1 gene (GST)P1 gene. Methods Using methylation -special PCR (MSP), the methylated status of CpG islands of GSTP1 gene in tumor tissues of 53 HCC and its adjacent nontumor tissues were studied. The en-

  20. Studying Genes

    Science.gov (United States)

    ... NIGMS NIGMS Home > Science Education > Studying Genes Studying Genes Tagline (Optional) Middle/Main Content Area Other Fact Sheets What are genes? Genes are segments of DNA that contain instructions ...

  1. Correlation of gene expression and contaminat concentrations in wild largescale suckers: a field-based study

    Science.gov (United States)

    Christiansen, Helena E.; Mehinto, Alvine C.; Yu, Fahong; Perry, Russell W.; Denslow, Nancy D.; Maule, Alec G.; Mesa, Matthew G.

    2014-01-01

    Toxic compounds such as organochlorine pesticides (OCs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether flame retardants (PBDEs) have been detected in fish, birds, and aquatic mammals that live in the Columbia River or use food resources from within the river. We developed a custom microarray for largescale suckers (Catostomus macrocheilus) and used it to investigate the molecular effects of contaminant exposure on wild fish in the Columbia River. Using Significance Analysis of Microarrays (SAM) we identified 72 probes representing 69 unique genes with expression patterns that correlated with hepatic tissue levels of OCs, PCBs, or PBDEs. These genes were involved in many biological processes previously shown to respond to contaminant exposure, including drug and lipid metabolism, apoptosis, cellular transport, oxidative stress, and cellular chaperone function. The relation between gene expression and contaminant concentration suggests that these genes may respond to environmental contaminant exposure and are promising candidates for further field and laboratory studies to develop biomarkers for monitoring exposure of wild fish to contaminant mixtures found in the Columbia River Basin. The array developed in this study could also be a useful tool for studies involving endangered sucker species and other sucker species used in contaminant research.

  2. Gene expression-based biological test for major depressive disorder: an advanced study

    Science.gov (United States)

    Watanabe, Shin-ya; Numata, Shusuke; Iga, Jun-ichi; Kinoshita, Makoto; Umehara, Hidehiro; Ishii, Kazuo; Ohmori, Tetsuro

    2017-01-01

    Purpose Recently, we could distinguished patients with major depressive disorder (MDD) from nonpsychiatric controls with high accuracy using a panel of five gene expression markers (ARHGAP24, HDAC5, PDGFC, PRNP, and SLC6A4) in leukocyte. In the present study, we examined whether this biological test is able to discriminate patients with MDD from those without MDD, including those with schizophrenia and bipolar disorder. Patients and methods We measured messenger ribonucleic acid expression levels of the aforementioned five genes in peripheral leukocytes in 17 patients with schizophrenia and 36 patients with bipolar disorder using quantitative real-time polymerase chain reaction (PCR), and we combined these expression data with our previous expression data of 25 patients with MDD and 25 controls. Subsequently, a linear discriminant function was developed for use in discriminating between patients with MDD and without MDD. Results This expression panel was able to segregate patients with MDD from those without MDD with a sensitivity and specificity of 64% and 67.9%, respectively. Conclusion Further research to identify MDD-specific markers is needed to improve the performance of this biological test. PMID:28260899

  3. Gene expression-based biological test for major depressive disorder: an advanced study

    Directory of Open Access Journals (Sweden)

    Watanabe S

    2017-02-01

    Full Text Available Shin-ya Watanabe,1 Shusuke Numata,1 Jun-ichi Iga,2 Makoto Kinoshita,1 Hidehiro Umehara,1 Kazuo Ishii,3 Tetsuro Ohmori1 1Department of Psychiatry, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, 2Department of Neuropsychiatry, Molecules and Function, Ehime University Graduate School of Medicine, Ehime, 3Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan Purpose: Recently, we could distinguished patients with major depressive disorder (MDD from nonpsychiatric controls with high accuracy using a panel of five gene expression markers (ARHGAP24, HDAC5, PDGFC, PRNP, and SLC6A4 in leukocyte. In the present study, we examined whether this biological test is able to discriminate patients with MDD from those without MDD, including those with schizophrenia and bipolar disorder.Patients and methods: We measured messenger ribonucleic acid expression levels of the aforementioned five genes in peripheral leukocytes in 17 patients with schizophrenia and 36 patients with bipolar disorder using quantitative real-time polymerase chain reaction (PCR, and we combined these expression data with our previous expression data of 25 patients with MDD and 25 controls. Subsequently, a linear discriminant function was developed for use in discriminating between patients with MDD and without MDD.Results: This expression panel was able to segregate patients with MDD from those without MDD with a sensitivity and specificity of 64% and 67.9%, respectively.Conclusion: Further research to identify MDD-specific markers is needed to improve the performance of this biological test. Keywords: depressive disorder, biomarker, gene expression, schizophrenia, bipolar disorder

  4. Cloning and expression study of the lobster (Homarus americanus) vitellogenin: Conservation in gene structure among decapods.

    Science.gov (United States)

    Tiu, Shirley Hiu Kwan; Hui, Ho-Lam; Tsukimura, Brian; Tobe, Stephen S; He, Jian-Guo; Chan, Siu-Ming

    2009-01-01

    This study reports the molecular characterization of the vitellogenin (Vg) of the lobster, Homarus americanus. Based on the annual collection of female lobsters, vitellogenesis commences in early March and continues through to September of each year. Using an antibody to vitellin of the lobster, H. americanus, several immunoreactive ovarian proteins were initially identified by Western blot analysis. The 80kDa protein contained the amino acid sequence APWGGNTPRC, identified subsequently by cDNA cloning to be identical to the lobster Vg. In common with the shrimp Metapenaeus ensis and crab Charybdis feriatus, the lobster HaVg1 gene comprises 14 introns and 15 exons. The deduced HaVg1 precursor is most similar to the Vg of the crayfish Cherax quadricarinatus (57%), followed by M. ensis (40-43% identity) and C. feriatus (38%). The results from genomic and RT-PCR cloning also confirmed the presence of multiple Vg genes in lobster. At early reproductive stages, the hepatopancreas HaVg1 transcript levels are low but increased to a maximum in animals with mature oocytes. The ovary, however, also expressed low levels of HaVg1. Using in vitro explant culture, treatment of hepatopancreas fragments with farnesoic acid or 20-hydroxyecdysone resulted in a significant stimulation in HaVg1 expression. From this study, it appears that Vg gene organization and expression pattern in decapods is highly conserved. Similar endocrine mechanisms may govern the process of vitellogenesis across the decapods.

  5. Analysis of Saliva Gene Expression during Head and Neck Cancer Radiotherapy: A Pilot Study.

    Science.gov (United States)

    Lacombe, Jerome; Brooks, Carla; Hu, Chengcheng; Menashi, Emmanuel; Korn, Ronald; Yang, Farley; Zenhausern, Frederic

    2017-07-01

    Saliva, a biological fluid, is a promising candidate for novel approaches to prognosis, clinical diagnosis, monitoring and management of patients with both oral and systemic diseases. However, to date, saliva has not been widely investigated as a biomarker for radiation exposure. Since white blood cells are also present in saliva, it should theoretically be possible to investigate the transcriptional biomarkers of radiation exposure classically studied in whole blood. Therefore, we collected whole blood and saliva samples from eight head and neck cancer patients before the start of radiation treatment, at mid-treatment and after treatment. We then used a panel of five genes: BAX, BBC3, CDKN1A, DDB2 and MDM2, designated for assessing radiation dose in whole blood to evaluate gene expression changes that can occur during radiotherapy. The results revealed that the expression of the five genes did not change in whole blood. However, in saliva, CDKN1A and DDB2 were significantly overexpressed at the end, compared to the start, of radiotherapy, and MDM2 was significantly underexpressed between mid-treatment and at the end of treatment. Interestingly, CDKN1A and DDB2 expressions also showed an increasing monotonic relationship with total radiation dose received during radiotherapy. To our knowledge, these results show for the first time the ability to detect gene expression changes in saliva after head and neck cancer radiotherapy, and pave the way for further promising studies validating saliva as a minimally invasive means of biofluid collection to directly measure radiation dose escalation during treatment.

  6. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    Science.gov (United States)

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  7. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    Science.gov (United States)

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  8. Evaluation and comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants

    NARCIS (Netherlands)

    Ruijter, de N.C.A.; Verhees, J.A.; Leeuwen, van W.; Krol, van der A.R.

    2003-01-01

    The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like beta-glucuronidase (GUS), green fluor

  9. Studies on gene expressions analyses for Arabidopsis thaliana plants stimulated by space flight condition

    Science.gov (United States)

    Lu, Jinying; Liu, Min; Pan, Yi; Li, Huasheng

    We carried out whole-genome microarray to screen the transcript profile of Arabidopsis thaliana seedlings after three treatment: space microgravity condition( Seedlings grown in microgravity state of space flight of SIMBOX on Shenzhou-8), 1g centrifugal force in space(Seedlings grown in 1g centrifugal force state of space flight of SIMBOX on Shenzhou-8) and ground control. The result of microarray analysis is as followed: There were 368 genes significantly differentially expressed in space microgravity condition compared with that in 1g centrifuge space condition. Space radiation caused 246 genes significantly differentially expressed between seedlings in 1g centrifuge space condition and ground control. Space conditions (including microgravity and radiation) caused 621 genes significantly differentially expressed between seedlings in space microgravity condition and ground control. Microgravity and radiation as a single factor can cause plant gene expression change, but two factors synergism can produce some new effects on plant gene expression. The function of differential expression genes were analyst by bioinformatics, and we found the expression of genes related with stress were more different, such as the dehydration of protein (dehydrin Xero2) expression is up-regulated 57 times; low-temperature-induced protein expression is up-regulated in 49 times; heat shock protein expression is up-regulated 20 times; transcription factor DREB2A expression increase 25 times; protein phosphatase 2C expression is up-regulated 14 times; transcription factor NAM-like protein expression is up-regulated 13 times; cell wall metabolism related genes (xyloglucan, endo-1, 4-beta-D-glucanase) expression is down-regulated in 15 times. The results provide scientific data for the mechanism of space mutation.

  10. A pipeline to determine RT-QPCR control genes for evolutionary studies: application to primate gene expression across multiple tissues.

    Directory of Open Access Journals (Sweden)

    Olivier Fedrigo

    Full Text Available Because many species-specific phenotypic differences are assumed to be caused by differential regulation of gene expression, many recent investigations have focused on measuring transcript abundance. Despite the availability of high-throughput platforms, quantitative real-time polymerase chain reaction (RT-QPCR is often the method of choice because of its low cost and wider dynamic range. However, the accuracy of this technique heavily relies on the use of multiple valid control genes for normalization. We created a pipeline for choosing genes potentially useful as RT-QPCR control genes for measuring expression between human and chimpanzee samples across multiple tissues, using published microarrays and a measure of tissue-specificity. We identified 13 genes from the pipeline and from commonly used control genes: ACTB, USP49, ARGHGEF2, GSK3A, TBP, SDHA, EIF2B2, GPDH, YWHAZ, HPTR1, RPL13A, HMBS, and EEF2. We then tested these candidate genes and validated their expression stability across species. We established the rank order of the most preferable set of genes for single and combined tissues. Our results suggest that for at least three tissues (cerebral cortex, liver, and skeletal muscle, EIF2B2, EEF2, HMBS, and SDHA are useful genes for normalizing human and chimpanzee expression using RT-QPCR. Interestingly, other commonly used control genes, including TBP, GAPDH, and, especially ACTB do not perform as well. This pipeline could be easily adapted to other species for which expression data exist, providing taxonomically appropriate control genes for comparisons of gene expression among species.

  11. A combined strategy of "in silico" transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies.

    Science.gov (United States)

    Faccioli, Primetta; Ciceri, Gian Paolo; Provero, Paolo; Stanca, Antonio Michele; Morcia, Caterina; Terzi, Valeria

    2007-03-01

    Traditionally housekeeping genes have been employed as endogenous reference (internal control) genes for normalization in gene expression studies. Since the utilization of single housekeepers cannot assure an unbiased result, new normalization methods involving multiple housekeeping genes and normalizing using their mean expression have been recently proposed. Moreover, since a gold standard gene suitable for every experimental condition does not exist, it is also necessary to validate the expression stability of every putative control gene on the specific requirements of the planned experiment. As a consequence, finding a good set of reference genes is for sure a non-trivial problem requiring quite a lot of lab-based experimental testing. In this work we identified novel candidate barley reference genes suitable for normalization in gene expression studies. An advanced web search approach aimed to collect, from publicly available web resources, the most interesting information regarding the expression profiling of candidate housekeepers on a specific experimental basis has been set up and applied, as an example, on stress conditions. A complementary lab-based analysis has been carried out to verify the expression profile of the selected genes in different tissues and during heat shock response. This combined dry/wet approach can be applied to any species and physiological condition of interest and can be considered very helpful to identify putative reference genes to be shortlisted every time a new experimental design has to be set up.

  12. Wetlab-2 - Quantitative PCR Tools for Spaceflight Studies of Gene Expression Aboard the International Space Station

    Science.gov (United States)

    Schonfeld, Julie E.

    2015-01-01

    Wetlab-2 is a research platform for conducting real-time quantitative gene expression analysis aboard the International Space Station. The system enables spaceflight genomic studies involving a wide variety of biospecimen types in the unique microgravity environment of space. Currently, gene expression analyses of space flown biospecimens must be conducted post flight after living cultures or frozen or chemically fixed samples are returned to Earth from the space station. Post-flight analysis is limited for several reasons. First, changes in gene expression can be transient, changing over a timescale of minutes. The delay between sampling on Earth can range from days to months, and RNA may degrade during this period of time, even in fixed or frozen samples. Second, living organisms that return to Earth may quickly re-adapt to terrestrial conditions. Third, forces exerted on samples during reentry and return to Earth may affect results. Lastly, follow up experiments designed in response to post-flight results must wait for a new flight opportunity to be tested.

  13. Epigenetic phenomena, chromatin dynamics, and gene expression. New theoretical approaches in the study of living systems.

    Science.gov (United States)

    Boi, Luciano

    2008-01-01

    This paper is aimed at exploring the genome at the level beyond that of DNA sequence alone. We stress the fact that the level of genes is not the sole "reality" in the living world, for there are different epigenetic processes that profoundly affect change in living systems. Moreover, epigenetics very likely influences the course of evolution and the unfolding of life. We further attempt to investigate how the genome is dynamically organized into the nuclear space within the cell. We mainly focus on analyses of higher order nuclear architecture and the dynamic interactions of chromatin with other nuclear components. We especially want to know how epigenetic phenomena influences genes expression and chromosome functions. The proper understanding of these processes require new concepts and approaches be introduced and developed. In particular, we think that research in biology has to shift from only describing molecular and local features of living systems to studying the regulatory networks of interactions among gene pathways, the folding and dynamics of chromatin structure and how environmental factors affects the behavior of organisms. There are essential components of biological information on living organisms which cannot be portrayed in the DNA sequence alone. In a post-genomic era, the importance of chromatin/epigenetic interface has become increasingly apparent. One of the purposes of current research should be to highlight the enormous impact of chromatin organization and dynamics on epigenetic phenomena, and, conversely, to emphasize the important role that epigenetic phenomena play in gene expression and cell regulation.

  14. Experimental approaches for the study of oxytocin and vasopressin gene expression in the central nervous system

    Science.gov (United States)

    Scordalakes, Elka M.; Yue, Chunmei; Gainer, Harold

    2016-01-01

    Intron-specific probes measure heteronuclear RNA (hnRNA) levels and thus approximate the transcription rates of genes, in part because of the rapid turnover of this intermediate form of RNA in the cell nucleus. Previously, we used oxytocin (Oxt)- and vasopressin (Avp)- intron-specific riboprobes to measure changes in Oxt and Avp hnRNA levels in the supraoptic nucleus (SON) by quantitative in situ hybridization (ISH) after various classical physiological perturbations, including acute and chronic salt loading, and lactation. In the present experiments, we used a novel experimental model to study the neurotransmitter regulation of Oxt and Avp gene expression in the rat SON in vivo. Bilateral cannulae connected via tubing to Alzet osmotic mini-pumps were positioned over the SON. In every experiment, one SON was infused with PBS and served as the control SON in each animal, and the contralateral SON received infusions of various neurotransmitter agonists and antagonists. Using this approach, we found that Avp but not Oxt gene expression increased after acute (2–5 h) combined excitatory amino acid agonist and GABA antagonist treatment, similar to what we found after an acute hyperosmotic stimulus. Since both OXT and AVP are known to be comparably and robustly secreted in response to acute osmotic stimuli in vivo and glutamate agonists in vitro, our results indicate a dissociation between OXT secretion and Oxt gene transcription in vivo. PMID:18655870

  15. Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

    OpenAIRE

    Becker Sven; Scharsack Joern P; Hibbeler Sascha

    2008-01-01

    Abstract Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG), depending on the tissue and experimental treatmen...

  16. Prioritizing orphan proteins for further study using phylogenomics and gene expression profiles in Streptomyces coelicolor

    Directory of Open Access Journals (Sweden)

    Takano Eriko

    2011-09-01

    Full Text Available Abstract Background Streptomyces coelicolor, a model organism of antibiotic producing bacteria, has one of the largest genomes of the bacterial kingdom, including 7825 predicted protein coding genes. A large number of these genes, nearly 34%, are functionally orphan (hypothetical proteins with unknown function. However, in gene expression time course data, many of these functionally orphan genes show interesting expression patterns. Results In this paper, we analyzed all functionally orphan genes of Streptomyces coelicolor and identified a list of "high priority" orphans by combining gene expression analysis and additional phylogenetic information (i.e. the level of evolutionary conservation of each protein. Conclusions The prioritized orphan genes are promising candidates to be examined experimentally in the lab for further characterization of their function.

  17. Antisense expression increases gene expression variability and locus interdependency

    OpenAIRE

    Xu, Zhenyu; Wei, Wu; Gagneur, Julien; Clauder-Münster, Sandra; Smolik, Miłosz; Huber, Wolfgang; Steinmetz, Lars M.

    2011-01-01

    Genome-wide transcription profiling has revealed extensive expression of non-coding RNAs antisense to genes, yet their functions, if any, remain to be understood. In this study, we perform a systematic analysis of sense–antisense expression in response to genetic and environmental changes in yeast. We find that antisense expression is associated with genes of larger expression variability. This is characterized by more ‘switching off' at low levels of expression for genes with antisense compa...

  18. Gene expression throughout a vertebrate's embryogenesis

    Directory of Open Access Journals (Sweden)

    Hinton David E

    2011-02-01

    Full Text Available Abstract Background Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. Results Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases. Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. Conclusions These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development.

  19. Learning from microarray interlaboratory studies: measures of precision for gene expression

    Directory of Open Access Journals (Sweden)

    Reid Laura H

    2009-04-01

    Full Text Available Abstract Background The ability to demonstrate the reproducibility of gene expression microarray results is a critical consideration for the use of microarray technology in clinical applications. While studies have asserted that microarray data can be "highly reproducible" under given conditions, there is little ability to quantitatively compare amongst the various metrics and terminology used to characterize and express measurement performance. Use of standardized conceptual tools can greatly facilitate communication among the user, developer, and regulator stakeholders of the microarray community. While shaped by less highly multiplexed systems, measurement science (metrology is devoted to establishing a coherent and internationally recognized vocabulary and quantitative practice for the characterization of measurement processes. Results The two independent aspects of the metrological concept of "accuracy" are "trueness" (closeness of a measurement to an accepted reference value and "precision" (the closeness of measurement results to each other. A carefully designed collaborative study enables estimation of a variety of gene expression measurement precision metrics: repeatability, several flavors of intermediate precision, and reproducibility. The three 2004 Expression Analysis Pilot Proficiency Test collaborative studies, each with 13 to 16 participants, provide triplicate microarray measurements on each of two reference RNA pools. Using and modestly extending the consensus ISO 5725 documentary standard, we evaluate the metrological precision figures of merit for individual microarray signal measurement, building from calculations appropriate to single measurement processes, such as technical replicate expression values for individual probes on a microarray, to the estimation and display of precision functions representing all of the probes in a given platform. Conclusion With only modest extensions, the established metrological framework

  20. Bayesian hierarchical clustering for studying cancer gene expression data with unknown statistics.

    Directory of Open Access Journals (Sweden)

    Korsuk Sirinukunwattana

    Full Text Available Clustering analysis is an important tool in studying gene expression data. The Bayesian hierarchical clustering (BHC algorithm can automatically infer the number of clusters and uses Bayesian model selection to improve clustering quality. In this paper, we present an extension of the BHC algorithm. Our Gaussian BHC (GBHC algorithm represents data as a mixture of Gaussian distributions. It uses normal-gamma distribution as a conjugate prior on the mean and precision of each of the Gaussian components. We tested GBHC over 11 cancer and 3 synthetic datasets. The results on cancer datasets show that in sample clustering, GBHC on average produces a clustering partition that is more concordant with the ground truth than those obtained from other commonly used algorithms. Furthermore, GBHC frequently infers the number of clusters that is often close to the ground truth. In gene clustering, GBHC also produces a clustering partition that is more biologically plausible than several other state-of-the-art methods. This suggests GBHC as an alternative tool for studying gene expression data. The implementation of GBHC is available at https://sites.google.com/site/gaussianbhc/

  1. cDNA-SRAP and Its Application in Differential Gene Expression Analysis: A Case Study in Erianthus arundinaceum

    Directory of Open Access Journals (Sweden)

    Youxiong Que

    2012-01-01

    Full Text Available Erianthus arundinaceum is a wild relative species of sugarcane. The aim of this research was to demonstrate the feasibility of cDNA-SRAP for differential gene expression and to explore the molecular mechanism of drought resistance in E. arundinaceum. cDNA-SRAP technique, for the first time, was applied in the analysis of differential gene expression in E. arundinaceum under drought stress. In total, eight differentially expressed genes with length of 185–427 bp were successfully isolated (GenBank Accession numbers: EU071770, EU071772, EU071774, EU071776, EU071777, EU071779, EU071780, and EU071781. Based on their homologies with genes in GenBank, these genes were assumed to encode ribonuclease III, vacuolar protein, ethylene insensitive protein, aerobactin biosynthesis protein, photosystem II protein, glucose transporter, leucine-rich repeat protein, and ammonia monooxygenase. Real-time PCR analysis on the expression profiling of gene (EU071774 encoding ethylene-insensitive protein and gene (EU071781 encoding ammonia monooxygenase revealed that the expression of these two genes was upregulated both by PEG and ABA treatments, suggesting that they may involve in the drought resistance of E. arundinaceum. This study constitutes the first report of genes activated in E. arundinaceum by drought stress and opens up the application of cDNA-SRAP in differential gene expression analysis in E. arundinaceum under certain stress conditions.

  2. The amylose-free potato mutant as a model plant to study gene expression and gene silencing.

    NARCIS (Netherlands)

    Flipse, E.

    1995-01-01

    In this thesis, gene-expression and gene silencing were examined for Granule Bound Starch Synthase (GBSS) which catalyses the formation of amylose and Branching Enzyme (BE) which catalyses the formation of amylopectin. The (GBSS) deficient, with iodine, red staining amylose-free (amf) potato mutant

  3. Studies on Expression of P1 Protamine Gene in Rat and Mouse Testis

    Institute of Scientific and Technical Information of China (English)

    费仁仁; 纪林; 吴小芳; 陈晖; 陈惠琼; 李建国; 王一飞; 闫月敏; 王莎丽; 陈啸梅; 薛社普

    1999-01-01

    Protamine is a kind small,basic protein rich in arginine residues and found to be complexed with DNA in spermatozoa. We have cloned a 150 bp cDNA encoding the rat protamine (rP) by RT-PCR technique.Dig-labelled cDNA for rP was used for Northern blot analysis to study the expression of P1 protamine gene in rat and mouse.P1 protamine mRNA was detected only in rat testis,no hybridization signals were de-tected in rat brain and lever.In addition,the presence of P1 protamine mRNA was detected not only in rat testis,but also in mouse testis.Dig-labelled cDNA for mouse protamine 1 (mP1) was used to study the expression of mP1 gene during the process of sexual maturation of mouse.7-8 d after birth,no mP1 mRNA could be detected.At d 24-26,mP1 mRNA was detectable migrating as a homogeneous band at 580 nu-cleotides,whereas in sexually mature animals,a heterogeneous mixture of RNAs ranging from 450-580 bases in length was observed.Histological studies revealed that in the testis of 7-8-day-old mouse, spermatogenesis has developed to the sperma-tocyte stage, whereas round spermatids (Rs) were present in the testis of the mice with 24-26 d age and elongating spermatids(Es)were present in the testis of sexually mature animals. Electrophoresis of total nuclear basic proteins(TNBP)revealed that the Rs could possess the somatic histones,while Es was found to have protamine and less histone.These results indicate that the P1 protamine gene is tissues-specifically ex-pressed and the P1 protamine is showing to be conservative in evolution.During the process of sexual maturation,along with morphological changes,mP1 gene was tran-scribed in Rs and translated in Es.The mechanism of protamine gene expression was discussed.

  4. Using oocyte nuclei for studies on chromatin structure and gene expression.

    Science.gov (United States)

    Sommerville, John

    2010-05-01

    The giant nucleus of amphibian oocytes is generally referred to as the germinal vesicle (GV). Its size allows relatively easy manual isolation from the rest of the oocyte and also presents a large target in situ for microinjection of macromolecules including plasmid DNA, RNA species, antibodies and other proteins and even whole organelles, including somatic cell nuclei. Thus the use of GVs is excellent for two major types of study: the function of endogenous nuclear processes such as gene transcription, RNA processing and intra-nuclear dynamics; and the use of the nuclear components to effect processes such as chromatin assembly, expression of foreign genes and nucleocytoplasmic transport of injected biomolecules. This article outlines some basic techniques appropriate for GV studies, particularly the preparation of oocytes for microinjection and the isolation of germinal vesicles into an oil phase. As an aid to the targeting of the GV within the nucleus, descriptions are given of the use of oocytes from albino animals.

  5. COMPARATIVE STUDY OF FIBRONECTIN GENE EXPRESSION IN TISSUES FROM HYPERTROPHIC SCARS AND DIABETIC FOOT ULCERS

    Institute of Scientific and Technical Information of China (English)

    付小兵; 杨银辉; 孙同柱; 王亚平; 盛志勇

    2002-01-01

    Objective.To explore the expression characteristic of fibronectin gene in hypertrophic scars and diabetic ulcer tissues.Methods.The biopsies from normal skins,hypertrophic scars and diabetic foot ulcers were taken.The technique of quantitative polymerase chain reaction (PCR) was used to evaluate the gene expression of fibronectin in the above biopsies.Results.Fibronectin gene expression was enhanced in hypertrophic scars and decreased in diabetic foot ulcers compared with that in normal skins.Quantitative comparison showed about 2 fold increase of fibronectin mRNA level in hypertrophic scars and about 3 fold decrease of fibronectin mRNA level in diabetic ulcers as compared with that in normal skins.Conclusions.Fibronectin gene expression is influenced by the tissue environment.Different expression and synthesis of fibronectin may cause different outcomes in wound healing.

  6. Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

    Directory of Open Access Journals (Sweden)

    Pitzschke Andrea

    2012-05-01

    Full Text Available Abstract Background Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue. Results Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70% of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima. In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days. Conclusions The simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications.

  7. Study on the relationship between the RAR-β gene expressive defection and its methylation

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To observe the expression of RAR-β gene in SiHa, HeLa,C33A and CasKi cell lines of cervical carcinoma and to investigate the role of methylated RAR-β in its expressive defection. Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of RAR-β gene. Immunohistochemistry and Western Blot were used to analyze the protein expression of RAR-β gene in four cervical cancer cell lines as well as the influence of 5-Aza-cdR on gene expressive defection. Methylation specific PCR (MSP) was used to detect whether there was the methylation in RAR-β gene in four cell lines. The change of RAR-β gene methylation state was also observed by MSP. The cell proliferation rate influenced by the 5-Aza-cdR was observed by MTT assay. Results The expression of RAR-β mRNA and protein in SiHa, HeLa and CasKi cell lines of cervical cancer was silent or decreased, whereas its expression was detected in C33A cell line. By using MSP method, it was found that there was RAR-β gene methylation in those three cell lines, whereas there was no RAR-β gene methylation in C33A cell line. After treated with the 5-Aza-cdR, methylated RAR-β gene was partly demethylated, and RAR-β mRNA and protein were re-expressed in the previous three cell lines in which RAR-β gene expression was silent or decreased. The 5-Aza-cdR treatment could supress cell proliferation as well. Conclusion The RAR-β gene expressive defection plays an important role in the carcinogenesis of cervical cancer. The abnormal RAR-β gene methylation in the the promotor region has an important role in gene expressive defection. The cell proliferation can be supressed by demethylated treatment.

  8. Optimization of an animal test protocol for toxicogenomics studies (ii); a cross-laboratory gene expression analysis.

    Science.gov (United States)

    Sumida, Kayo; Saito, Koichi; Oeda, Kenji; Otsuka, Masanori; Tsujimura, Kazunari; Miyaura, Hideki; Sekijima, Masaru; Nakayama, Koji; Kawano, Yukiko; Kawakami, Yuki; Asamoto, Makoto; Shirai, Tomoyuki

    2007-02-01

    Toxicogenomics is a promising new tool for prediction of chemical toxicities including carcinogenicity in a relatively short period. However, it is important to develop a reliable animal test protocol for toxicogenomics studies. The preparation of RNA and tissues is also crucial, since it greatly influences outcomes of gene expression analysis. We proposed an animal test protocol for toxicogenomics studies. In the present study, we examined an animal test protocol by comparing biological and gene expression data from different laboratories running identical in vivo studies on the same microarray platform. The results gave good correspondence in all three laboratories at the level of biological responses and gene expression, especially for genes whose expression changes were quite large. As the fold change or the signal values become smaller, however, discrepancies occur in gene expression data. For example, one laboratory shows an opposite directional change to the other two or no change. The results of hierarchical clustering and principal component analysis (PCA) demonstrated all samples from the three laboratories being clearly divided between control and treatment. Examination of the reproducibility of gene expression data across laboratories using the proposed animal test protocol thus confirmed only minor differences, which was expected to present no problems for gene expression analysis.

  9. Microarray studies on effects of Pneumocystis carinii infection on global gene expression in alveolar macrophages

    Directory of Open Access Journals (Sweden)

    Liao Chung-Ping

    2010-04-01

    Full Text Available Abstract Background Pneumocystis pneumonia is a common opportunistic disease in AIDS patients. The alveolar macrophage is an important effector cell in the clearance of Pneumocystis organisms by phagocytosis. However, both the number and phagocytic activity of alveolar macrophages are decreased in Pneumocystis infected hosts. To understand how Pneumocystis inactivates alveolar macrophages, Affymetrix GeneChip® RG-U34A DNA microarrays were used to study the difference in global gene expression in alveolar macrophages from uninfected and Pneumocystis carinii-infected Sprague-Dawley rats. Results Analyses of genes that were affected by Pneumocystis infection showed that many functions in the cells were affected. Antigen presentation, cell-mediated immune response, humoral immune response, and inflammatory response were most severely affected, followed by cellular movement, immune cell trafficking, immunological disease, cell-to-cell signaling and interaction, cell death, organ injury and abnormality, cell signaling, infectious disease, small molecular biochemistry, antimicrobial response, and free radical scavenging. Since rats must be immunosuppressed in order to develop Pneumocystis infection, alveolar macrophages from four rats of the same sex and age that were treated with dexamethasone for the entire eight weeks of the study period were also examined. With a filter of false-discovery rate less than 0.1 and fold change greater than 1.5, 200 genes were found to be up-regulated, and 144 genes were down-regulated by dexamethasone treatment. During Pneumocystis pneumonia, 115 genes were found to be up- and 137 were down-regulated with the same filtering criteria. The top ten genes up-regulated by Pneumocystis infection were Cxcl10, Spp1, S100A9, Rsad2, S100A8, Nos2, RT1-Bb, Lcn2, RT1-Db1, and Srgn with fold changes ranging between 12.33 and 5.34; and the top ten down-regulated ones were Lgals1, Psat1, Tbc1d23, Gsta1, Car5b, Xrcc5, Pdlim1, Alcam

  10. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each p...... with a high frequency of loss of heterozygosity. The genes and ESTs presented in this study encode new potential tumor markers as well as potential novel therapeutic targets for prevention or therapy of CRC.......Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226...

  11. Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

    Science.gov (United States)

    Varshney, N; Mohanty, A K; Kumar, S; Kaushik, J K; Dang, A K; Mukesh, M; Mishra, B P; Kataria, R; Kimothi, S P; Mukhopadhyay, T K; Malakar, D; Prakash, B S; Grover, S; Batish, V K

    2012-06-01

    We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), β-actin (ACTB), β-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, β-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

  12. [Study on gene expression of Tamarix under NaHCO3 stress using SSH technology].

    Science.gov (United States)

    Yang, Chuan-Ping; Wang, Yu-Cheng; Liu, Gui-Feng; Jiang, Jing

    2004-09-01

    The gene expression of Tamarix androssowii under NaHCO3 stresses is studied by using SSH technique, in which the cDNA from the materials treated with NaHCO3 solution is as tester and the cDNA from the materials in normal growth is as driver. Total 36 genes related to NaHCO3 stress were obtained through Northern hybridization. Blastx analysis showed that the proteins encoded by these genes were homologous to the following proteins: the antioxidant enzymes catalase and peroxiredoxin; trehalose phosphatase, which was related to trehalose synthesis; a few regulation proteins such as bZIP transcription factor, MADS-box protein, glycine-rich RNA-binding proteins, CCCH-type zinc finger protein and F-box protein etc; early light-induced protein, which could protect and/or repair the photosynthetic apparatus damage induced by stress; cysteine proteinase and vacuolar processing enzyme that can make function in plant cell death, and lipid transfer protein precursor, polyubiquitin, chalcone synthase, NADP-dependent isocitrate dehydrogenase, salt-induced S12 protein, and oxygen-evolving enhancer protein 1 etc. Among 36 genes obtained, the proteins encoded three genes were homologous to 3 putative proteins: HAK2, calcium-binding protein and RNA-binding protein, respectively. In addition, 6 new salt stress response squences were found. The result indicated that the salt-tolerant mechanism of Tamarix androssowii may be a complicated, interactive system involving multiple approaches and multiple genes, but not only a single salt gland-depended approach.

  13. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin;

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies...... an analytical approach to examine the suitability of correction methods by considering the inter-treatment bias as well as the inter-replicate variance, which allows use of the best correction method with minimum residual bias. Analyses of RNA sequencing and microarray data showed that the efficiencies...

  14. Common handling procedures conducted in preclinical safety studies result in minimal hepatic gene expression changes in Sprague-Dawley rats.

    Directory of Open Access Journals (Sweden)

    Yudong D He

    Full Text Available Gene expression profiling is a tool to gain mechanistic understanding of adverse effects in response to compound exposure. However, little is known about how the common handling procedures of experimental animals during a preclinical study alter baseline gene expression. We report gene expression changes in the livers of female Sprague-Dawley rats following common handling procedures. Baseline gene expression changes identified in this study provide insight on how these changes may affect interpretation of gene expression profiles following compound exposure. Rats were divided into three groups. One group was not subjected to handling procedures and served as controls for both handled groups. Animals in the other two groups were weighed, subjected to restraint in Broome restrainers, and administered water via oral gavage daily for 1 or 4 days with tail vein blood collections at 1, 2, 4, and 8 hours postdose on days 1 and 4. Significantly altered genes were identified in livers of animals following 1 or 4 days of handling when compared to the unhandled animals. Gene changes in animals handled for 4 days were similar to those handled for 1 day, suggesting a lack of habituation. The altered genes were primarily immune function related genes. These findings, along with a correlating increase in corticosterone levels suggest that common handling procedures may cause a minor immune system perturbance.

  15. Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon

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    Xu Cheng

    2012-11-01

    Full Text Available Abstract Salmonid alphavirus subtype-3 (SAV-3 infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM, cohabited (CO or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi. Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs of both IM and CO groups, with changes appearing first in the pancreas (2 wpi in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.

  16. Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon.

    Science.gov (United States)

    Xu, Cheng; Guo, Tz-Chun; Mutoloki, Stephen; Haugland, Oyvind; Evensen, Oystein

    2012-11-01

    Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.

  17. A developmental biological study of aldolase gene expression in Xenopus laevis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.

  18. Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca [v1; ref status: indexed, http://f1000r.es/y1

    Directory of Open Access Journals (Sweden)

    Teresa Docimo

    2013-02-01

    Full Text Available Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves, and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca (Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3. The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper. From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced, while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.

  19. Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

    Science.gov (United States)

    Lau, Yun-Fai; Kan, Yuet Wai

    1983-09-01

    We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

  20. Quantitative studies of DNA methylation and gene expression in neuropsychiatric traits

    NARCIS (Netherlands)

    van Eijk, K.R.

    2014-01-01

    Research has shown that besides genes and environment, epigenetic factors are also playing a role in the development of traits and diseases. Epigenetic changes do not affect the underlying DNA sequence, but affect its structure and is thought to play a major role in regulation of gene expression. DN

  1. To what extent is blood a reasonable surrogate for brain in gene expression studies: estimation from mouse hippocampus and spleen

    Directory of Open Access Journals (Sweden)

    Matthew N Davies

    2009-10-01

    Full Text Available Microarrays are designed to measure genome-wide differences in gene expression. In cases where a tissue is not accessible for analysis (e.g. human brain, it is of interest to determine whether a second, accessible tissue could be used as a surrogate for transcription profiling. Surrogacy has applications in the study of behavioural and neurodegenerative disorders. Comparison between hippocampus and spleen mRNA obtained from a mouse recombinant inbred panel indicates a high degree of correlation between the tissues for genes that display a high heritability of expression level. This correlation is not limited to apparent expression differences caused by sequence polymorphisms in the target sequences and includes both cis and trans genetic effects. A tissue such as blood could therefore give surrogate information on expression in brain for a subset of genes, in particular those co-expressed between the two tissues, which have heritably varying expression.

  2. To What Extent is Blood a Reasonable Surrogate for Brain in Gene Expression Studies: Estimation from Mouse Hippocampus and Spleen.

    Science.gov (United States)

    Davies, Matthew N; Lawn, Sarah; Whatley, Steven; Fernandes, Cathy; Williams, Robert W; Schalkwyk, Leonard C

    2009-01-01

    Microarrays are designed to measure genome-wide differences in gene expression. In cases where a tissue is not accessible for analysis (e.g. human brain), it is of interest to determine whether a second, accessible tissue could be used as a surrogate for transcription profiling. Surrogacy has applications in the study of behavioural and neurodegenerative disorders. Comparison between hippocampus and spleen mRNA obtained from a mouse recombinant inbred panel indicates a high degree of correlation between the tissues for genes that display a high heritability of expression level. This correlation is not limited to apparent expression differences caused by sequence polymorphisms in the target sequences and includes both cis and trans genetic effects. A tissue such as blood could therefore give surrogate information on expression in brain for a subset of genes, in particular those co-expressed between the two tissues, which have heritably varying expression.

  3. Autoradiography study and SPECT imaging of reporter gene HSV1-tk expression in heart

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: LXL730724@hotmail.com; Liu Ying; He Yong; Wu Tao; Zhang Binqing; Gao Zairong; An Rui [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, Hubei Province, 430022 (China)], E-mail: zhyx1229@163.com

    2010-04-15

    Aim: To demonstrate the feasibility and optimal conditions of imaging herpes simplex virus 1-thymidine kinase (HSV1-tk) gene transferred into hearts with {sup 131}I-2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil ({sup 131}I-FIAU) using autoradiography (ARG) and single photon emission computed tomography (SPECT) in animal models. Methods: HSV1-tk inserted into adenovirus vector (Ad5-tk) and adenovirus (Ad5-null) was prepared. Rats or rabbits were divided into a study group receiving intramyocardial injection of Ad5-tk, and a control group receiving Ad-null injection. In the study group of rats, two sets of experiments, time-course study and dose-dependence study, were performed. In time-course experiments, rats were injected with {sup 131}I-FIAU on Days 1, 2, 3, 5 and 7, after transfection of 1x10{sup 8} pfu Ad5-tk, to study the feasibility and suitable time course for reporter gene imaging. In dose-dependence study, various titers of Ad5-tk (5x10{sup 8}, 1x10{sup 8}, 5x10{sup 7} and 1x10{sup 7} pfu) were used to determine the threshold and optimal viral titer needed for detection of gene expression. The gamma counts of hearts were measured. The rat myocardium was analyzed by ARG and reverse transcriptase-polymerase chain reaction (RT-PCR). SPECT whole-body planar imaging and cardiac tomographic imaging were performed in the rabbit models. Results: From the ARG images, rats injected with Ad5-tk showed significant {sup 131}I-FIAU activity in the anterolateral wall compared with background signals seen in the control Ad5-null rats. In time-course study, the highest radioactivity in the focal myocardium could be seen on Day 1, and then progressively declined with time. In dose-dependence study, the level of {sup 131}I-FIAU accumulation in the transfected myocardium declined with the decrease of Ad viral titers. From the ARG analysis and gamma counting, the threshold viral titer was 5x10{sup 7} pfu, and the optimal Ad titer was 1x10{sup 8} pfu

  4. Selection of Reference Genes for Expression Study in Pulp and Seeds of Theobroma grandiflorum (Willd. ex Spreng.) Schum

    Science.gov (United States)

    Ferraz dos Santos, Lucas; Santana Silva, Raner José; Oliveira Jordão do Amaral, Daniel; Barbosa de Paula, Márcia Fabiana; Ludke Falcão, Loeni; Legavre, Thierry; Alves, Rafael Moyses

    2016-01-01

    Cupuassu (Theobroma grandiflorum [Willd. ex Spreng.] Schum) is a species of high economic importance in Brazil with great potential at international level due to the multiple uses of both its seeds and pulp in the industry of sweets and cosmetics. For this reason, the cupuassu breeding program focused on the selection of genotypes with high pulp and seed quality—selection associated with the understanding of the mechanisms involved in fruit formation. Gene expression is one of the most used approaches related to such understanding. In this sense, quantitative real-time PCR (qPCR) is a powerful tool, since it rapidly and reliably quantifies gene expression levels across different experimental conditions. The analysis by qPCR and the correct interpretation of data depend on signal normalization using reference genes, i.e. genes presenting a uniform pattern of expression in the analyzed samples. Here, we selected and analyzed the expression of five genes from cupuassu (ACP, ACT, GAPDH, MDH, TUB) to be used as candidates for reference genes on pulp and seed of young, maturing and mature cupuassu fruits. The evaluation of the gene expression stability was obtained using the NormFinder, geNorm and BestKeeper programs. In general, our results indicated that the GAPDH and MDH genes constituted the best combination as reference genes to analyze the expression of cupuassu samples. To our knowledge, this is the first report of reference gene definition in cupuassu, and these results will support subsequent analysis related to gene expression studies in cupuassu plants subjected to different biotic or abiotic conditions as well as serve as a tool for diversity analysis based on pulp and seed quality. PMID:27501324

  5. MRI of Transgene Expression: Correlation to Therapeutic Gene Expression

    Directory of Open Access Journals (Sweden)

    Tomotsugu Ichikawa

    2002-01-01

    Full Text Available Magnetic resonance imaging (MRI can provide highresolution 3D maps of structural and functional information, yet its use of mapping in vivo gene expression has only recently been explored. A potential application for this technology is to noninvasively image transgene expression. The current study explores the latter using a nonregulatable internalizing engineered transferrin receptor (ETR whose expression can be probed for with a superparamagnetic Tf-CLIO probe. Using an HSV-based amplicon vector system for transgene delivery, we demonstrate that: 1 ETR is a sensitive MR marker gene; 2 several transgenes can be efficiently expressed from a single amplicon; 3 expression of each transgene results in functional gene product; and 4 ETR gene expression correlates with expression of therapeutic genes when the latter are contained within the same amplicon. These data, taken together, suggest that MRI of ETR expression can serve as a surrogate for measuring therapeutic transgene expression.

  6. Heterologous expression and activity studying of a nisin resistance gene in Lactobacillus delbrueckii subsp. bulgaricus

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    Fanghong Gong

    2013-04-01

    Full Text Available Background: Lactic acid bacteria are generally regarded as safe microorganisms and widely used in industry and medicine. We are trying to add additional properties to them by gene engineering. However, the genetically modified bacteria are not acceptable to use in food and medicine due to the presence of antibiotic resistance genes in plasmids. Thus, it is necessary to develop food-grade selection markers. Methods: To evaluate the activity of nisin resistance gene (nsr cloned from Lactococcus lactis as a food-grade selective marker in transformant bacterial strains, we isolated L.lactis nisin resistant strains from fresh milk, and cloned the nsr gene to Lactobacillus delbrueckii subsp. bulgaricus, then studied the activity of nsr in positive transformant bacterial strains. Results: Six nisin resistant strains were isolated from fresh milk on M17 plates supplemented with nisin at a final concentration of 400 μg/ml. All of these isolates were identified as L.lactis by morphological, physiological, biochemical and 16S rDNA sequence analysis. A pair of primers was designed on the basis of the DNA sequences of reported nsr. PCR amplification was carried out with chromosome and plasmid DNA as templates from these six strains respectively, and an expected PCR product was obtained from one chromosome. After the amplicon was confirmed as nsr gene, it was cloned into E.coli-Lactobacillus shuttle vector pMG36e, resulting in pMG36e-nsr. The construct was obtained when the recombinant plasmid pMG36e-nsr was electroporated into L.delbrueckii subsp. bulgaricus L6032 competent cells. When the medium contained a maximum of 400 μg/ml nisin, the construct carrying pMG36e-nsr showed the same growth curve as that cultured in medium containing 10 μg/ml erythromycin. Conclusions: The nsr gene was cloned and successfully expressed in L.delbrueckii subsp. bulgaricus L6032. This study suggests that nsr gene could be used as a potential food-grade selective marker to

  7. Adaptive Evolution of Gene Expression in Drosophila

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    Armita Nourmohammad

    2017-08-01

    Full Text Available Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis.

  8. Tumor-specific gene expression patterns with gene expression profiles

    Institute of Scientific and Technical Information of China (English)

    RUAN Xiaogang; LI Yingxin; LI Jiangeng; GONG Daoxiong; WANG Jinlian

    2006-01-01

    Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.

  9. The effect of oxythioquinox exposure on normal human mammary epithelial cell gene expression: A microarray analysis study

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    Weston Ainsley

    2004-09-01

    Full Text Available Abstract Background Inter-individual variation in normal human mammary epithelial cells in response to oxythioquinox (OTQ is reported. Gene expression signatures resulting from chemical exposures are generally created from analysis of exposures in rat, mouse or other genetically similar animal models, limiting information about inter-individual variations. This study focused on the effect of inter-individual variation in gene expression signatures. Methods Gene expression was studied in primary normal human mammary epithelial cells (NHMECs derived from four women undergoing reduction mammoplasty [Cooperative Human Tissue Network (National Cancer Institute and National Disease Research Interchange]. Gene transcription in each cell strain was analyzed using high-density oligonucleotide DNA microarrays (HuGeneFL, Affymetrix™ and changes in the expression of selected genes were verified by real-time polymerase chain reaction at extended time points (ABI. DNA microarrays were hybridized to materials prepared from total RNA that was collected after OTQ treatment for 15, 60 and 120 min. RNA was harvested from the vehicle control (DMSO at 120 min. The gene expression profile included all genes altered by at least a signal log ratio (SLR of ± 0.6 and p value ≤ 0.05 in three of four cell strains analyzed. Results RNA species were clustered in various patterns of expression highlighting genes with altered expression in one or more of the cell strains, including metabolic enzymes and transcription factors. Of the clustered RNA species, only 36 were found to be altered at one time point in three or more of the cell strains analyzed (13 up-regulated, 23 down-regulated. Cluster analysis examined the effects of OTQ on the cells with specific p53 polymorphisms. The two strains expressing the major variant of p53 had 83 common genes altered (35 increased, 48 decreased at one or more time point by at least a 0.6 signal log ratio (SLR. The intermediate variant

  10. Genome-wide gene expression study indicates the anti-inflammatory effect of polarized light in recurrent childhood respiratory disease.

    Science.gov (United States)

    Falus, A; Fenyo, M; Éder, K; Madarasi, A

    2011-10-01

    The clinical and molecular effects of whole-body polarized light treatment on children suffering from recurrent respiratory infection were studied. The incidence and duration of respiratory symptoms as well as the length of appropriate antibiotic therapy were measured. Simultaneously, the genome-wide gene expression pattern was examined by whole genome cDNA microarray in peripheral lymphocytes of children. Twenty of 25 children showed a marked clinical improvement, while in five of 25 had poor response or no changes. The gene expression pattern of the patients' peripheral lymphocytes was compared in favorable and poor responders. The lymphocytes of the children with a documented improved clinical response to polarized light therapy showed a decrease in the expression of chemokine genes, such as CXCL1, CXCL2, CXCL3, and IL-8, and in that of the TNFα gene. On the contrary, a rapid elevation was found in the expression of the gene encoding for CYP4F2, a leukotriene B4-metabolizing enzyme. In children with poor clinical response to polarized light therapy, no similar changes were detected in the gene expression pattern of the lymphocytes. The improved clinical symptoms and modified gene expression profile of lymphocytes reveals an anti-inflammatory effect of whole-body polarized light irradiation.

  11. Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

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    Toth Ian K

    2007-09-01

    Full Text Available Abstract Background Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and in planta experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen Pectobacterium atrosepticum, belonging to the family Enterobacteriaceae. Results Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and in planta. Two of these genes (recA and ffh, encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes proC and gyrA, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels. Conclusion Based on these results, we suggest recA and ffh as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen P. atrosepticum and potentially other related pathogens.

  12. Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR

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    Deforce Dieter

    2006-08-01

    Full Text Available Abstract Background Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence gene family can be performed using quantitative PCR (qPCR. In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans biofilms, using the geNorm Visual Basic Application (VBA for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts. Results The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable as follows: ACT1 (β-actin/PMA1 (adenosine triphosphatase, RIP (ubiquinol cytochrome-c reductase complex component, RPP2B (cytosolic ribosomal acidic protein P2B, LSC2 (succinyl-CoA synthetase β-subunit fragment, IMH3 (inosine-5'-monophosphate dehydrogenase fragment, CPA1 (carbamoyl-phosphate synthethase small subunit and GAPDH (glyceraldehyde-3-phosphate dehydrogenase. Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control, for 24 hours. Conclusion In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2 for normalization, when analysing differences in gene expression levels between C. albicans biofilm

  13. Predator-induced defences in Daphnia pulex: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

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    Gilbert Don

    2010-06-01

    Full Text Available Abstract Background The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data. Results In this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, TATA box binding protein (Tbp syntaxin 16 (Stx16, X-box binding protein 1 (Xbp1 and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP, was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are

  14. Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR

    Institute of Scientific and Technical Information of China (English)

    LI Bing; CHEN Changsheng; XU Yan; JI Dehua; XIE Chaotian

    2014-01-01

    Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expres-sion studies using quantitative real-time PCR should start by selecting an appropriate internal control gene;therefore, the absolute expression abundance of six housekeeping genes (18S rRNA (18S), ubiquitin-conju-gating enzyme (UBC), actin (ACT),β-tubulin (TUB), elongation factors 2 (EF2), and glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold (Ct) method and by two different software packages:geNorm and Norm-Finder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.

  15. Benzodiazepines use and breast cancer risk: A population-based study and gene expression profiling evidence.

    Science.gov (United States)

    Iqbal, Usman; Chang, Tzu-Hao; Nguyen, Phung-Anh; Syed-Abdul, Shabbir; Yang, Hsuan-Chia; Huang, Chih-Wei; Atique, Suleman; Yang, Wei-Chung; Moldovan, Max; Jian, Wen-Shan; Hsu, Min-Huei; Yen, Yun; Jack Li, Yu-Chuan

    2017-08-26

    The aim of this study was to investigate whether long-term use of Benzodiazepines (BZDs) is associated with breast cancer risk through the combination of population-based observational and gene expression profiling evidence. We conducted a population-based case-control study by using 1998 to 2009 year Taiwan National Health Insurance Research Database and investigated the association between BZDs use and breast cancer risk. We selected subjects age of > 20 years old and six eligible controls matched for age, sex and the index date (i.e., free of any cancer at the case diagnosis date) by using propensity scores. A bioinformatics analysis approach was also performed for the identification of oncogenesis effects of BZDs on breast cancer. We used breast cancer gene expression data from the Cancer Genome Atlas and perturbagen signatures of BZDs from the Library of Integrated Cellular Signatures database in order to identify the oncogenesis effects of BZDs on breast cancer. We found evidence of increased breast cancer risk for diazepam (OR, 1.16; 95%CI, 0.95-1.42; connectivity score [CS], 0.3016), zolpidem (OR, 1.11; 95%CI, 0.95-1.30; CS, 0.2738), but not for lorazepam (OR, 1.04; 95%CI, 0.89-1.23; CS, -0.2952) consistently in both methods. The finding for alparazolam was contradictory from the two methods. Diazepam and zolpidem trends showed association, although not statistically significant, with breast cancer risk in both epidemiological and bioinformatics analyses outcomes. The methodological value of our study is in introducing the way of combining epidemiological and bioinformatics approaches in order to answer a common scientific question. Combining the two approaches would be a substantial step towards uncovering, validation and further application of previously unknown scientific knowledge to the emerging field of precision medicine informatics. Copyright © 2017. Published by Elsevier Inc.

  16. Gene Expression Patterns in Peripheral Blood Leukocytes in Patients with Recurrent Ciguatera Fish Poisoning: Preliminary Studies.

    Science.gov (United States)

    Lopez, Maria-Cecilia; Ungaro, Ricardo F; Baker, Henry V; Moldawer, Lyle L; Robertson, Alison; Abbott, Margaret; Roberts, Sparkle M; Grattan, Lynn M; Morris, J Glenn

    2016-07-01

    Ciguatera fish poisoning (ciguatera) is a common clinical syndrome in areas where there is dependence on tropical reef fish for food. A subset of patients develops recurrent and, in some instances, chronic symptoms, which may result in substantial disability. To identify possible biomarkers for recurrent/chronic disease, and to explore correlations with immune gene expression, peripheral blood leukocyte gene expression in 10 ciguatera patients (7 recurrent, 3 acute) from the U.S. Virgin Islands, and 5 unexposed Florida controls were evaluated. Significant differences in gene expression were noted when comparing ciguatera patients and controls; however, it was not possible to differentiate between patients with acute and recurrent disease, possibly due to the small sample sizes involved.

  17. Examining Radiation-Induced In Vivo and In Vitro Gene Expression Changes of the Peripheral Blood in Different Laboratories for Biodosimetry Purposes: First RENEB Gene Expression Study.

    Science.gov (United States)

    Abend, M; Badie, C; Quintens, R; Kriehuber, R; Manning, G; Macaeva, E; Njima, M; Oskamp, D; Strunz, S; Moertl, S; Doucha-Senf, S; Dahlke, S; Menzel, J; Port, M

    2016-02-01

    The risk of a large-scale event leading to acute radiation exposure necessitates the development of high-throughput methods for providing rapid individual dose estimates. Our work addresses three goals, which align with the directive of the European Union's Realizing the European Network of Biodosimetry project (EU-RENB): 1. To examine the suitability of different gene expression platforms for biodosimetry purposes; 2. To perform this examination using blood samples collected from prostate cancer patients (in vivo) and from healthy donors (in vitro); and 3. To compare radiation-induced gene expression changes of the in vivo with in vitro blood samples. For the in vitro part of this study, EDTA-treated whole blood was irradiated immediately after venipuncture using single X-ray doses (1 Gy/min(-1) dose rate, 100 keV). Blood samples used to generate calibration curves as well as 10 coded (blinded) samples (0-4 Gy dose range) were incubated for 24 h in vitro, lysed and shipped on wet ice. For the in vivo part of the study PAXgene tubes were used and peripheral blood (2.5 ml) was collected from prostate cancer patients before and 24 h after the first fractionated 2 Gy dose of localized radiotherapy to the pelvis [linear accelerator (LINAC), 580 MU/min, exposure 1-1.5 min]. Assays were run in each laboratory according to locally established protocols using either microarray platforms (2 laboratories) or qRT-PCR (2 laboratories). Report times on dose estimates were documented. The mean absolute difference of estimated doses relative to the true doses (Gy) were calculated. Doses were also merged into binary categories reflecting aspects of clinical/diagnostic relevance. For the in vitro part of the study, the earliest report time on dose estimates was 7 h for qRT-PCR and 35 h for microarrays. Methodological variance of gene expression measurements (CV ≤10% for technical replicates) and interindividual variance (≤twofold for all genes) were low. Dose estimates based on

  18. Identification of reliable reference genes for quantitative gene expression studies in oral squamous cell carcinomas compared to adjacent normal tissues in the F344 rat model.

    Science.gov (United States)

    Peng, Xinjian; McCormick, David L

    2016-08-01

    Oral squamous cell carcinomas (OSCCs) induced in F344 rats by 4-nitroquinoline-1-oxide (4-NQO) demonstrate considerable phenotypic similarity to human oral cancers and the model has been widely used for carcinogenesis and chemoprevention studies. Molecular characterization of this model needs reliable reference genes (RGs) to avoid false- positive and -negative results for proper interpretation of gene expression data between tumor and adjacent normal tissues. Microarray analysis of 11 pairs of OSCC and site-matched phenotypically normal oral tissues from 4-NQO-treated rats identified 10 stably expressed genes in OSCC compared to adjacent normal tissues (p>0.5, CVexpression analysis. We successfully identified Hsp90ab1 as a stable RG in 4-NQO-induced OSCC compared to adjacent normal tissues in F344 rats. The combination of two stably expressed genes may be a better option for gene normalization in tissue samples.

  19. Study of five novel non-synonymous polymorphisms in human brain-expressed genes in a Colombian sample.

    Science.gov (United States)

    Ojeda, Diego A; Forero, Diego A

    2014-10-01

    Non-synonymous single nucleotide polymorphisms (nsSNPs) in brain-expressed genes represent interesting candidates for genetic research in neuropsychiatric disorders. To study novel nsSNPs in brain-expressed genes in a sample of Colombian subjects. We applied an approach based on in silico mining of available genomic data to identify and select novel nsSNPs in brain-expressed genes. We developed novel genotyping assays, based in allele-specific PCR methods, for these nsSNPs and genotyped them in 171 Colombian subjects. Five common nsSNPs (rs6855837; p.Leu395Ile, rs2305160; p.Thr394Ala, rs10503929; p.Met289Thr, rs2270641; p.Thr4Pro and rs3822659; p.Ser735Ala) were studied, located in the CLOCK, NPAS2, NRG1, SLC18A1 and WWC1 genes. We reported allele and genotype frequencies in a sample of South American healthy subjects. There is previous experimental evidence, arising from genome-wide expression and association studies, for the involvement of these genes in several neuropsychiatric disorders and endophenotypes, such as schizophrenia, mood disorders or memory performance. Frequencies for these nsSNPSs in the Colombian samples varied in comparison to different HapMap populations. Future study of these nsSNPs in brain-expressed genes, a synaptogenomics approach, will be important for a better understanding of neuropsychiatric diseases and endophenotypes in different populations.

  20. A comparative study of P450 gene expression in field and laboratory Musca domestica L. strains

    DEFF Research Database (Denmark)

    Højland, Dorte Heidi; Vagn Jensen, Karl-Martin; Kristensen, Michael

    2014-01-01

    BACKGROUND The housefly is a global pest that has developed resistance to most insecticides applied for its control. Resistance has been associated with cytochrome P450 monooxygenases (P450s). The authors compare the expression of six genes possibly associated with insecticide resistance in three...... in the multiresistant strain (P metabolic resistance. There is a strong indication...... that CYP6G4 is a major insecticide resistance gene involved in neonicotinoid resistance. © 2013 Society of Chemical Industry...

  1. Correlation of gene expression and protein production rate - a system wide study

    Directory of Open Access Journals (Sweden)

    Arvas Mikko

    2011-12-01

    Full Text Available Abstract Background Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype. Results We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR. We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response. Conclusions Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR.

  2. Genes involved in the osteoarthritis process identified through genome wide expression analysis in articular cartilage; the RAAK study.

    Directory of Open Access Journals (Sweden)

    Yolande F M Ramos

    Full Text Available Identify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process.Genome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions.Among the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. TNFRSF11B and FRZB. Also several inflammatory genes such as CD55, PTGES and TNFAIP6, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of NGF in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score but were independent of joint-site or sex.We provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways.

  3. Study of gene function based on spatial co-expression in a high-resolution mouse brain atlas

    Directory of Open Access Journals (Sweden)

    Jiang Tao

    2007-04-01

    Full Text Available Abstract Background The Allen Brain Atlas (ABA project systematically profiles three-dimensional high-resolution gene expression in postnatal mouse brains for thousands of genes. By unveiling gene behaviors at both the cellular and molecular levels, ABA is becoming a unique and comprehensive neuroscience data source for decoding enigmatic biological processes in the brain. Given the unprecedented volume and complexity of the in situ hybridization image data, data mining in this area is extremely challenging. Currently, the ABA database mainly serves as an online reference for visual inspection of individual genes; the underlying rich information of this large data set is yet to be explored by novel computational tools. In this proof-of-concept study, we studied the hypothesis that genes sharing similar three-dimensional expression profiles in the mouse brain are likely to share similar biological functions. Results In order to address the pattern comparison challenge when analyzing the ABA database, we developed a robust image filtering method, dubbed histogram-row-column (HRC algorithm. We demonstrated how the HRC algorithm offers the sensitivity of identifying a manageable number of gene pairs based on automatic pattern searching from an original large brain image collection. This tool enables us to quickly identify genes of similar in situ hybridization patterns in a semi-automatic fashion and consequently allows us to discover several gene expression patterns with expression neighborhoods containing genes of similar functional categories. Conclusion Given a query brain image, HRC is a fully automated algorithm that is able to quickly mine vast number of brain images and identify a manageable subset of genes that potentially shares similar spatial co-distribution patterns for further visual inspection. A three-dimensional in situ hybridization pattern, if statistically significant, could serve as a fingerprint of certain gene function

  4. A Growth Curve Model with Fractional Polynomials for Analysing Incomplete Time-Course Data in Microarray Gene Expression Studies

    Directory of Open Access Journals (Sweden)

    Qihua Tan

    2011-01-01

    Full Text Available Identifying the various gene expression response patterns is a challenging issue in expression microarray time-course experiments. Due to heterogeneity in the regulatory reaction among thousands of genes tested, it is impossible to manually characterize a parametric form for each of the time-course pattern in a gene by gene manner. We introduce a growth curve model with fractional polynomials to automatically capture the various time-dependent expression patterns and meanwhile efficiently handle missing values due to incomplete observations. For each gene, our procedure compares the performances among fractional polynomial models with power terms from a set of fixed values that offer a wide range of curve shapes and suggests a best fitting model. After a limited simulation study, the model has been applied to our human in vivo irritated epidermis data with missing observations to investigate time-dependent transcriptional responses to a chemical irritant. Our method was able to identify the various nonlinear time-course expression trajectories. The integration of growth curves with fractional polynomials provides a flexible way to model different time-course patterns together with model selection and significant gene identification strategies that can be applied in microarray-based time-course gene expression experiments with missing observations.

  5. Gene Expression in Trypanosomatid Parasites

    Directory of Open Access Journals (Sweden)

    Santiago Martínez-Calvillo

    2010-01-01

    Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

  6. Validation of suitable reference genes for expression studies in different pilocarpine-induced models of mesial temporal lobe epilepsy.

    Directory of Open Access Journals (Sweden)

    Thalita Ewellyn Batista Sales Marques

    Full Text Available It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

  7. Assessment of Control Tissue for Gene and Protein Expression Studies: A Comparison of Three Alternative Lung Sources

    Directory of Open Access Journals (Sweden)

    Margaret R. Passmore

    2012-01-01

    Full Text Available The use of an appropriate control group in human research is essential in investigating the level of a pathological disorder. This study aimed to compare three alternative sources of control lung tissue and to determine their suitability for gene and protein expression studies. Gene and protein expression levels of the vascular endothelial growth factor (VEGF and gelatinase families and their receptors were measured using real-time reverse transcription polymerase chain reaction (RT-PCR and immunohistochemistry. The gene expression levels of VEGFA, placental growth factor (PGF, and their receptors, fms-related tyrosine kinase 1 (FLT1, and kinase insert domain receptor (KDR as well as matrix metalloproteinase-2 (MMP-2 and the inhibitors, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1 and TIMP-2 were significantly higher in lung cancer resections. The gene expression level of MMP-9 was significantly lower in the corresponding samples. Altered protein expression was also detected, depending on the area assessed. The results of this study show that none of the three control groups studied are completely suitable for gene and protein studies associated with the VEGF and gelatinase families, highlighting the need for researchers to be selective in which controls they opt for.

  8. Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua l. during temperature stress

    Directory of Open Access Journals (Sweden)

    Karlsen Hans E

    2011-04-01

    Full Text Available Abstract Background One important physiological response to environmental stress in animals is change in gene expression. To obtain reliable data from gene expression studies using RT-qPCR it is important to evaluate a set of possible reference genes as normalizers for expression. The expression of these candidate genes should be analyzed in the relevant tissues during normal and stressed situations. To find suitable reference genes it was crucial that the genes were stably expressed also during a situation of physiological stress. For poikilotermic animals like cod, changes in temperature are normal, but if the changes are faster than physiological compensation, the animals respond with typical stress responses. It has previously been shown that Atlantic cod show stress responses when elevation of water temperature is faster than 1 degree/day, for this reason we chose hyperthermia as stress agent for this experiment. Findings We here describe the expression of eight candidate reference genes from Atlantic cod (Gadus morhua l. and their stability during thermal stress (temperature elevation of one degree C/day for 5 days. The genes investigated were: Eukaryotic elongation factor 1 alpha, ef1a; 18s ribosomal RNA; 18s, Ubiquitin conjugate protein; ubiq, cytoskeletal beta-actin; actb, major histcompatibility complex I; MHC-I light chain, beta-2 -microglobulin; b2m, cytoskeletal alpha-tubulin; tba1c, acidic ribosomal phosphoprotein; rplp1, glucose-6-phosphate dehydrogenase; g6pd. Their expression were analyzed in 6 tissues (liver, head kidney, intestine, spleen, heart and gills from cods exposed to elevated temperature and compared to a control group. Although there were variations between tissues with respect to reference gene stability, four transcripts were more consistent than the others: ubiq, ef1a, 18s and rplp1. We therefore used these to analyze the expression of stress related genes (heat shock proteins induced during hyperthermia. We found

  9. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Cirera, Susanna

    2007-01-01

    Background: Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high...... quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. Results: In this study nine commonly used reference genes were investigated in 17 different pig tissues using real-time qPCR with SYBR green. The genes included beta-actin (ACTB), beta-2...

  10. Selection of Reliable Reference Genes for Gene Expression Studies in the Biofuel Plant Jatropha curcas Using Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Lu Zhang

    2013-12-01

    Full Text Available Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.

  11. Studies on the expression of plasmid-borne genes in the endosymbiotic state of Rhizobium leguminosarum

    NARCIS (Netherlands)

    Krol, A.J.M.

    1982-01-01

    The subject matter of the research reported in this thesis is the role of plasmid-borne genes of Rhizobium in symbiosis and nitrogen fixation. Plasmid DNA was isolated from Rhizobium leguminosarum strain PRE and the expression of plasmid DNA in nitrogen fixing nodules was investigated by hybridizati

  12. Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

    NARCIS (Netherlands)

    Hibbeler, S.; Scharsack, J.P.; Becker, S.

    2008-01-01

    Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the

  13. Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

    NARCIS (Netherlands)

    Hibbeler, S.; Scharsack, J.P.; Becker, S.

    2008-01-01

    Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the

  14. Expression and functional studies of ubiquitin C-terminal hydrolase L1 regulated genes.

    Directory of Open Access Journals (Sweden)

    Anjali Bheda

    Full Text Available Deubiquitinating enzymes (DUBs have been increasingly implicated in regulation of cellular processes, but a functional role for Ubiquitin C-terminal Hydrolases (UCHs, which has been largely relegated to processing of small ubiquitinated peptides, remains unexplored. One member of the UCH family, UCH L1, is expressed in a number of malignancies suggesting that this DUB might be involved in oncogenic processes, and increased expression and activity of UCH L1 have been detected in EBV-immortalized cell lines. Here we present an analysis of genes regulated by UCH L1 shown by microarray profiles obtained from cells in which expression of the gene was inhibited by RNAi. Microarray data were verified with subsequent real-time PCR analysis. We found that inhibition of UCH L1 activates genes that control apoptosis, cell cycle arrest and at the same time suppresses expression of genes involved in proliferation and migration pathways. These findings are complemented by biological assays for apoptosis, cell cycle progression and migration that support the data obtained from microarray analysis, and suggest that the multi-functional molecule UCH L1 plays a role in regulating principal pathways involved in oncogenesis.

  15. cis sequence effects on gene expression

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    Jacobs Kevin

    2007-08-01

    Full Text Available Abstract Background Sequence and transcriptional variability within and between individuals are typically studied independently. The joint analysis of sequence and gene expression variation (genetical genomics provides insight into the role of linked sequence variation in the regulation of gene expression. We investigated the role of sequence variation in cis on gene expression (cis sequence effects in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. We estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature. Results We generated gene expression profiling data at N = 697 candidate genes from N = 30 lymphoblastoid cell lines for this study and used available candidate gene resequencing data at N = 552 candidate genes to identify N = 30 candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. We used two additive models and the haplotype phylogeny scanning approach of Templeton (Tree Scanning to evaluate association between individual SNPs, all SNPs at a gene, and diplotypes, with log-transformed gene expression. SNPs and diplotypes at eight candidate genes exhibited statistically significant (p cis sequence effects in our study, respectively. Conclusion Based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. Despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies.

  16. Selection of housekeeping genes for gene expression studies in the adult rat submandibular gland under normal, inflamed, atrophic and regenerative states

    Directory of Open Access Journals (Sweden)

    Osailan Samira

    2008-07-01

    Full Text Available Abstract Background Real-time PCR is a reliable tool with which to measure mRNA transcripts, and provides valuable information on gene expression profiles. Endogenous controls such as housekeeping genes are used to normalise mRNA levels between samples for sensitive comparisons of mRNA transcription. Selection of the most stable control gene(s is therefore critical for the reliable interpretation of gene expression data. For the purpose of this study, 7 commonly used housekeeping genes were investigated in salivary submandibular glands under normal, inflamed, atrophic and regenerative states. Results The program NormFinder identified the suitability of HPRT to use as a single gene for normalisation within the normal, inflamed and regenerative states, and GAPDH in the atrophic state. For normalisation to multiple housekeeping genes, for each individual state, the optimal number of housekeeping genes as given by geNorm was: ACTB/UBC in the normal, ACTB/YWHAZ in the inflamed, ACTB/HPRT in the atrophic and ACTB/GAPDH in the regenerative state. The most stable housekeeping gene identified between states (compared to normal was UBC. However, ACTB, identified as one of the most stably expressed genes within states, was found to be one of the most variable between states. Furthermore we demonstrated that normalising between states to ACTB, rather than UBC, introduced an approximately 3 fold magnitude of error. Conclusion Using NormFinder, our studies demonstrated the suitability of HPRT to use as a single gene for normalisation within the normal, inflamed and regenerative groups and GAPDH in the atrophic group. However, if normalising to multiple housekeeping genes, we recommend normalising to those identified by geNorm. For normalisation across the physiological states, we recommend the use of UBC.

  17. Brucella melitensis global gene expression study provides novel information on growth phase-specific gene regulation with potential insights for understanding Brucella:host initial interactions

    Directory of Open Access Journals (Sweden)

    Garner Harold R

    2009-05-01

    Full Text Available Abstract Background Brucella spp. are the etiological agents of brucellosis, a zoonotic infectious disease that causes abortion in animals and chronic debilitating illness in humans. Natural Brucella infections occur primarily through an incompletely defined mechanism of adhesion to and penetration of mucosal epithelium. In this study, we characterized changes in genome-wide transcript abundance of the most and the least invasive growth phases of B. melitensis cultures to HeLa cells, as a preliminary approach for identifying candidate pathogen genes involved in invasion of epithelial cells. Results B. melitensis at the late logarithmic phase of growth are more invasive to HeLa cells than mid-logarithmic or stationary growth phases. Microarray analysis of B. melitensis gene expression identified 414 up- and 40 down-regulated genes in late-log growth phase (the most invasive culture compared to the stationary growth phase (the least invasive culture. As expected, the majority of up-regulated genes in late-log phase cultures were those associated with growth, including DNA replication, transcription, translation, intermediate metabolism, energy production and conversion, membrane transport, and biogenesis of the cell envelope and outer membrane; while the down-regulated genes were distributed among several functional categories. Conclusion This Brucella global expression profile study provides novel information on growth phase-specific gene expression. Further characterization of some genes found differentially expressed in the most invasive culture will likely bring new insights into the initial molecular interactions between Brucella and its host.

  18. Study of the SH3-donain GRB2-like 2 gene expression in laryngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    SHANG Chao; FU Wei-neng; GUO Yan; HUANG Dai-fa; SUN Kai-lai

    2007-01-01

    Background Laryngeal carcinoma is a common malignant tumor of the upper respiratory tract, and in 95% of cases the tumor is laryngeal squamous cell carcinoma (LSCC). The abnormity of SH3-domain GRB2-like 2 (SH3GL2) gene was found in LSCC. In order to clarify the relationship between SH3GL2 gene and LSCC, we evaluated the expression of the SH3GL2 gene in LSCC.Method Real-time PCR, immunohistochemistry and Western blotting were used to detect the mRNA and protein expression and find the various rules of SH3GL2 gene in LSCC.Results The result of real-time PCR showed that the expression level of SH3GL2 mRNA in LSCC tissue was apparently down-regulated; immunohistochemical analysis showed that SH3GL2 protein was mainly located in cytoplasm, the rate of positive cells and SH3GL2 protein expression level were fluctuated with the pathological classification of LSCC; the result of Western blotting showed that SH3GL2 protein was down-regulated significantly in LSCC samples, especially in metastatic lymph nodes.Conclusions These results suggest that SH3GL2 is a LSCC related gene and its expression level is fluctuated with the pathological classification which indicate that SH3GL2 participates in the development and progression of LSCC. And it may be considered as a novel tumor marker to find both a new anti-oncogene and relative factors of invasion and metastasis of laryngeal carcinoma.

  19. Validation study of existing gene expression signatures for anti-TNF treatment in patients with rheumatoid arthritis.

    Directory of Open Access Journals (Sweden)

    Erik J M Toonen

    Full Text Available So far, there are no means of identifying rheumatoid arthritis (RA patients who will fail to respond to tumour necrosis factor blocking agents (anti-TNF, prior to treatment. We set out to validate eight previously reported gene expression signatures predicting therapy outcome. Genome-wide expression profiling using Affymetrix GeneChip Exon 1.0 ST arrays was performed on RNA isolated from whole blood of 42 RA patients starting treatment with infliximab or adalimumab. Clinical response according to EULAR criteria was determined at week 14 of therapy. Genes that have been reported to be associated with anti-TNF treatment were extracted from our dataset. K-means partition clustering was performed to assess the predictive value of the gene-sets. We performed a hypothesis-driven analysis of the dataset using eight existing gene sets predictive of anti-TNF treatment outcome. The set that performed best reached a sensitivity of 71% and a specificity of 61%, for classifying the patients in the current study. We successfully validated one of eight previously reported predictive expression profile. This replicated expression signature is a good starting point for developing a prediction model for anti-TNF treatment outcome that can be used in a daily clinical setting. Our results confirm that gene expression profiling prior to treatment is a useful tool to predict anti-TNF (non response.

  20. Validation study of existing gene expression signatures for anti-TNF treatment in patients with rheumatoid arthritis.

    Science.gov (United States)

    Toonen, Erik J M; Gilissen, Christian; Franke, Barbara; Kievit, Wietske; Eijsbouts, Agnes M; den Broeder, Alfons A; van Reijmersdal, Simon V; Veltman, Joris A; Scheffer, Hans; Radstake, Timothy R D J; van Riel, Piet L C M; Barrera, Pilar; Coenen, Marieke J H

    2012-01-01

    So far, there are no means of identifying rheumatoid arthritis (RA) patients who will fail to respond to tumour necrosis factor blocking agents (anti-TNF), prior to treatment. We set out to validate eight previously reported gene expression signatures predicting therapy outcome. Genome-wide expression profiling using Affymetrix GeneChip Exon 1.0 ST arrays was performed on RNA isolated from whole blood of 42 RA patients starting treatment with infliximab or adalimumab. Clinical response according to EULAR criteria was determined at week 14 of therapy. Genes that have been reported to be associated with anti-TNF treatment were extracted from our dataset. K-means partition clustering was performed to assess the predictive value of the gene-sets. We performed a hypothesis-driven analysis of the dataset using eight existing gene sets predictive of anti-TNF treatment outcome. The set that performed best reached a sensitivity of 71% and a specificity of 61%, for classifying the patients in the current study. We successfully validated one of eight previously reported predictive expression profile. This replicated expression signature is a good starting point for developing a prediction model for anti-TNF treatment outcome that can be used in a daily clinical setting. Our results confirm that gene expression profiling prior to treatment is a useful tool to predict anti-TNF (non) response.

  1. Correlations of gene expression with ratings of inattention and hyperactivity/impulsivity in tourette syndrome: a pilot study

    Directory of Open Access Journals (Sweden)

    Tian Yingfang

    2012-10-01

    Full Text Available Abstract Background Inattentiveness, impulsivity and hyperactivity are the primary behaviors associated with attention-deficit hyperactivity disorder (ADHD. Previous studies showed that peripheral blood gene expression signatures can mirror central nervous system disease. Tourette syndrome (TS is associated with inattention (IA and hyperactivity/impulsivity (HI symptoms over 50% of the time. This study determined if gene expression in blood correlated significantly with IA and/or HI rating scale scores in participants with TS. Methods RNA was isolated from the blood of 21 participants with TS, and gene expression measured on Affymetrix human U133 Plus 2.0 arrays. To identify the genes that correlated with Conners’ Parents Ratings of IA and HI ratings of symptoms, an analysis of covariance (ANCOVA was performed, controlling for age, gender and batch. Results There were 1201 gene probesets that correlated with IA scales, 1625 that correlated with HI scales, and 262 that correlated with both IA and HI scale scores (Prp|>0.4. Immune, catecholamine and other neurotransmitter pathways were associated with IA and HI behaviors. A number of the identified genes (n=27 have previously been reported in ADHD genetic studies. Many more genes correlated with either IA or HI scales alone compared to those that correlated with both IA and HI scales. Conclusions These findings support the concept that the pathophysiology of ADHD and/or its subtypes in TS may involve the interaction of multiple genes. These preliminary data also suggest gene expression may be useful for studying IA and HI symptoms that relate to ADHD in TS and perhaps non-TS participants. These results will need to be confirmed in future studies.

  2. The Gene Expression Omnibus database

    Science.gov (United States)

    Clough, Emily; Barrett, Tanya

    2016-01-01

    The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome–protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

  3. Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

    Science.gov (United States)

    Taylor, Candy M; Jost, Ricarda; Erskine, William; Nelson, Matthew N

    2016-01-01

    Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more

  4. Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L..

    Directory of Open Access Journals (Sweden)

    Candy M Taylor

    Full Text Available Quantitative Reverse Transcription PCR (qRT-PCR is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC, Helicase (HEL, and Polypyrimidine tract-binding protein (PTB] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other

  5. Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

    DEFF Research Database (Denmark)

    Jacobsen, Kari Stougaard; Nielsen, Kirstine Overgaard; Nordmann Winther, Thilde;

    2016-01-01

    BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially...... of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes...... identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model....

  6. Sample size matters in dietary gene expression studies—A case study in the gilthead sea bream (Sparus aurata L.

    Directory of Open Access Journals (Sweden)

    Fotini Kokou

    2016-05-01

    Full Text Available One of the main concerns in gene expression studies is the calculation of statistical significance which in most cases remains low due to limited sample size. Increasing biological replicates translates into more effective gains in power which, especially in nutritional experiments, is of great importance as individual variation of growth performance parameters and feed conversion is high. The present study investigates in the gilthead sea bream Sparus aurata, one of the most important Mediterranean aquaculture species. For 24 gilthead sea bream individuals (biological replicates the effects of gradual substitution of fish meal by plant ingredients (0% (control, 25%, 50% and 75% in the diets were studied by looking at expression levels of four immune-and stress-related genes in intestine, head kidney and liver. The present results showed that only the lowest substitution percentage is tolerated and that liver is the most sensitive tissue to detect gene expression variations in relation to fish meal substituted diets. Additionally the usage of three independent biological replicates were evaluated by calculating the averages of all possible triplets in order to assess the suitability of selected genes for stress indication as well as the impact of the experimental set up, thus in the present work the impact of FM substitution. Gene expression was altered depending of the selected biological triplicate. Only for two genes in liver (hsp70 and tgf significant differential expression was assured independently of the triplicates used. These results underlined the importance of choosing the adequate sample number especially when significant, but minor differences in gene expression levels are observed.

  7. Identification of novel GH-regulated pathway of lipid metabolism in adipose tissue: a gene expression study in hypopituitary men.

    Science.gov (United States)

    Zhao, Jing Ting; Cowley, Mark J; Lee, Paul; Birzniece, Vita; Kaplan, Warren; Ho, Ken K Y

    2011-07-01

    Adipose tissue is a major target of GH action. GH stimulates lipolysis and reduces fat mass. The molecular mechanism underlying cellular and metabolic effects of GH in adipose tissue is not well understood. The aim of this study is to identify GH-responsive genes that regulate lipid metabolism in adipose tissue. Eight men with GH deficiency underwent measurement of plasma free fatty acid (FFA), whole-body lipid oxidation, and fat biopsies before and after 1 month of GH treatment (0.5 mg/d). Gene expression profiling was performed using Agilent 44K G4112F arrays using a two-color design. Differentially expressed genes were identified using an empirical Bayes, moderated t test, with a false discovery rate under 5%. Target genes were validated by quantitative RT-PCR. GH increased circulating IGF-I and FFA and stimulated fat oxidation. A total of 246 genes were differentially expressed, of which 135 were up-regulated and 111 down-regulated. GH enhanced adipose tissue expression of IGF-I and SOCS3. GH increased expression of patatin-like phospholipase domain containing 3 (PNPLA3), a novel triglyceride (TG) hydrolase, but not hormone-sensitive lipase (HSL), a classical TG hydrolase. GH repressed cell death-inducing DFFA-like effector A (CIDEA), a novel lipid droplets-associated protein, promoting TG storage. GH differentially regulated genes promoting diacylglycerol synthesis. GH suppressed hydroxysteroid (11β) dehydrogenase 1, which activates local cortisol production and genes encoding components of extracellular matrix and TGF-β signaling pathway. GH stimulates the TG/FFA cycle by regulating the expression of novel genes that enhance TG hydrolysis, reduce TG storage, and promote diacylglycerol synthesis. GH represses adipocyte growth, differentiation and inflammation.

  8. Diagnostic value of blood gene expression signatures in active tuberculosis in Thais: a pilot study.

    Science.gov (United States)

    Satproedprai, N; Wichukchinda, N; Suphankong, S; Inunchot, W; Kuntima, T; Kumpeerasart, S; Wattanapokayakit, S; Nedsuwan, S; Yanai, H; Higuchi, K; Harada, N; Mahasirimongkol, S

    2015-06-01

    Tuberculosis (TB) is a major global health problem. Routine laboratory tests or newly developed molecular detection are limited to the quality of sputum sample. Here we selected genes specific to TB by a minimum redundancy-maximum relevancy package using publicly available microarray data and determine level of selected genes in blood collected from a Thai TB cohort of 40 active TB patients, 38 healthy controls and 18 previous TB patients using quantitative real-time PCR. FCGR1A, FCGR1B variant 1, FCGR1B variant 2, APOL1, GBP5, PSTPIP2, STAT1, KCNJ15, MAFB and KAZN had significantly higher expression level in active TB individuals as compared with healthy controls and previous TB cases (P<0.01). A mathematical method was applied to calculate TB predictive score, which contains the level of expression of seven genes and this score can identify active TB cases with 82.5% sensitivity and 100% specificity as compared with conventional culture confirmation. In addition, TB predictive scores in active TB patients were reduced to normal after completion of standard short-course therapy, which was mostly in concordant with the disease outcome. These finding suggested that blood gene expression measurement and TB Sick Score could have potential value in terms of diagnosis of TB and anti-TB treatment monitoring.

  9. Gene expression in response to exercise in patients with chronic fatigue syndrome: a pilot study.

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    Andrew Keech

    2016-09-01

    Full Text Available Chronic fatigue syndrome (CFS is a debilitating disorder of unknown pathogenesis, characterised by fatigue, which is exacerbated after minimal exercise. We examined the effect of a single bout of aerobic exercise on leucocyte mRNA expression of genes putatively linked to exaggerated afferent signalling as an under-pinning of the fatigue state. A carefully-characterised sample of patients with CFS (N = 10 and healthy matched control participants (N = 12 were included. Participant ratings of fatigue and other symptoms, as well as blood samples, were obtained at baseline, and five other time-points up to 72 hours after 25 minutes of moderate-intensity cycling exercise. Leucocyte mRNA of 19 metabolite-sensing, adrenergic, immune and neurotransmission genes was examined using quantitative polymerase chain reaction. Patients with CFS reported substantial fatigue, functional impairment and poor sleep at baseline (all p < 0.02, and exercise immediately induced worsened patients’ fatigue (effect size, ES = 1.17. There were no significant changes in gene expression after exercise and patients did not differ from control participants at any time point. Higher levels of expression of ficolin (FCN1 and a purinergic receptor (P2RX4 in patients with CFS were found when all time points were combined. Patients with CFS did not show significant exercise-induced changes in leucocyte mRNA of 19 metabolite-sensing, adrenergic, immune and neurotransmission genes despite a prominent exacerbation of fatigue.

  10. Combining gene expression, demographic and clinical data in modeling disease: a case study of bipolar disorder and schizophrenia

    Directory of Open Access Journals (Sweden)

    Dobrin Seth

    2008-11-01

    Full Text Available Abstract Background This paper presents a retrospective statistical study on the newly-released data set by the Stanley Neuropathology Consortium on gene expression in bipolar disorder and schizophrenia. This data set contains gene expression data as well as limited demographic and clinical data for each subject. Previous studies using statistical classification or machine learning algorithms have focused on gene expression data only. The present paper investigates if such techniques can benefit from including demographic and clinical data. Results We compare six classification algorithms: support vector machines (SVMs, nearest shrunken centroids, decision trees, ensemble of voters, naïve Bayes, and nearest neighbor. SVMs outperform the other algorithms. Using expression data only, they yield an area under the ROC curve of 0.92 for bipolar disorder versus control, and 0.91 for schizophrenia versus control. By including demographic and clinical data, classification performance improves to 0.97 and 0.94 respectively. Conclusion This paper demonstrates that SVMs can distinguish bipolar disorder and schizophrenia from normal control at a very high rate. Moreover, it shows that classification performance improves by including demographic and clinical data. We also found that some variables in this data set, such as alcohol and drug use, are strongly associated to the diseases. These variables may affect gene expression and make it more difficult to identify genes that are directly associated to the diseases. Stratification can correct for such variables, but we show that this reduces the power of the statistical methods.

  11. Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes.

    Science.gov (United States)

    Kar, Parmita; Chawla, Himika; Saha, Soma; Tandon, Nikhil; Goswami, Ravinder

    2016-06-01

    Selection of appropriate housekeeping-genes as reference is important in mRNA expression-related experiments. It is more important in diabetes since hyperglycemia per se can influence expression of housekeeping-genes. RNA expression of Glyceraldehyde-3-phosphate-dehydrogenase, β-actin and 18S-ribosomal-RNA, Hypoxanthine-phosphoribosyl-transferase (HPRT), Tyrosine-3-monooxygenase/tryptophan (YHWAZ), β2-microglobin (β2M), TATA-binding-protein (TBP), and Ubiquitin C and cytochrome1 (CYC1) assessed in circulating-lymphocytes-(PBMC) of patients with type-1-diabetes and healthy controls. The stability ('M' value genes required for normalization in qRT-PCR were determined by 'ge-norm software.' Vitamin-D-receptor (VDR) was used as a target gene. All the nine genes tested had sufficient 'M' value in diabetes and healthy controls. However, housekeeping-genes indicated a relatively higher stability of expression in healthy controls in comparison to diabetes. Use of single housekeeping-genes brought gross variation in the calculation of VDR-mRNA copies. The ge-norm software suggested geometric mean of five housekeeping-genes for ideal normalization in diabetes (CYC1, β-actin, YHWAZ, HPRT, and β2M) and only three in controls (CYC1, β-actin, and TBP). HbA1c did not correlate with expression of any of the nine housekeeping-genes. Thus, geometric mean of CYC1, β-actin, YHWAZ, HPRT, and β2M needs to be used for ideal normalization of mRNA in type-1-diabetes. Similar studies are required in other population.

  12. Use of the Chinchilla Model for Nasopharyngeal Colonization To Study Gene Expression by Moraxella catarrhalis

    Science.gov (United States)

    Hoopman, Todd C.; Liu, Wei; Joslin, Stephanie N.; Pybus, Christine; Sedillo, Jennifer L.; Labandeira-Rey, Maria; Laurence, Cassie A.; Wang, Wei; Richardson, James A.; Bakaletz, Lauren O.

    2012-01-01

    Young adult chinchillas were atraumatically inoculated with Moraxella catarrhalis via the nasal route. Detailed histopathologic examination of nasopharyngeal tissues isolated from these M. catarrhalis-infected animals revealed the presence of significant inflammation within the epithelium. Absence of similar histopathologic findings in sham-inoculated animals confirmed that M. catarrhalis was exposed to significant host-derived factors in this environment. Twenty-four hours after inoculation, viable M. catarrhalis organisms were recovered from the nasal cavity and nasopharynx of the animals in numbers sufficient for DNA microarray analysis. More than 100 M. catarrhalis genes were upregulated in vivo, including open reading frames (ORFs) encoding proteins that are involved in a truncated denitrification pathway or in the oxidative stress response, as well as several putative transcriptional regulators. Additionally, 200 M. catarrhalis genes were found to be downregulated when this bacterium was introduced into the nasopharynx. These downregulated genes included ORFs encoding several well-characterized M. catarrhalis surface proteins including Hag, McaP, and MchA1. Real-time reverse transcriptase PCR (RT-PCR) was utilized as a stringent control to validate the results of in vivo gene expression patterns as measured by DNA microarray analysis. Inactivation of one of the genes (MC ORF 1550) that was upregulated in vivo resulted in a decrease in the ability of M. catarrhalis to survive in the chinchilla nasopharynx over a 3-day period. This is the first evaluation of global transcriptome expression by M. catarrhalis cells in vivo. PMID:22184412

  13. Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR.

    Science.gov (United States)

    Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

    2016-01-01

    Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes.

  14. Neuronal gene expression in aluminum-induced neurofibrillary pathology: an in situ hybridization study.

    Science.gov (United States)

    Chambers, C B; Muma, N A

    1997-01-01

    Alterations in cytoskeletal proteins such as the perikaryal accumulation of neurofilaments (NFs) occur in a number of human neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis and may contribute to their debilitating effects. The administration of aluminum salts to rabbits induces the aberrant accumulation of NFs within the proximal axons and perikarya of vulnerable neurons and is one animal model which has been extensively studied in an attempt to gain insight into the mechanism(s) of NF perturbations in human disease. Previous studies using Northern blotting techniques to examine mRNA levels in the aluminum-induced neuropathy model have led to seemingly contradictory results. We have used in situ hybridization which provides the cellular resolution needed to: 1) determine whether there are generalized decreases in the levels of mRNA expression or decreases in mRNA encoding specific proteins; 2) determine whether alterations in mRNA levels occur specifically in neurons with NF accumulations; and 3) begin to resolve some of the apparent contradictions in the literature. A moderate dose of aluminum lactate administered on two consecutive days produced neurofibrillary tangles in spinal cord neurons seven days after the first dose. Polyadenylated mRNA levels were not altered in spinal cord neurons in aluminum-treated compared to saline-treated control animals or in tangle-bearing compared to non tangle-bearing neurons in aluminum-treated animals. Middle and high NF subunit (NFH) mRNA levels were not significantly different from polyadenylated mRNA levels in spinal cord neurons in aluminum-treated/control animals. NFH mRNA levels were decreased in neurons containing aluminum-induced NF accumulations. These results suggest that NFH gene expression may be down regulated by an inhibitory feedback mechanism induced by perikaryal accumulations of NFs. This inhibitory feedback regulation for NFH may have

  15. The roles of Tenascin C and Fibronectin 1 in adhesive capsulitis: a pilot gene expression study

    Directory of Open Access Journals (Sweden)

    Carina Cohen

    Full Text Available OBJECTIVES: We evaluated mRNA expression levels of genes that encode TGF-β1; the TGF-β1 receptor; the collagen-modifying enzymes LOX, PLOD1, and PLOD2; and the extracellular matrix proteins COMP, FN1, TNC and TNXB in synovial/capsule specimens from patients with idiopathic adhesive capsulitis. Possible associations between the measured mRNA levels and clinical parameters were also investigated. METHODS: We obtained glenohumeral joint synovium/capsule specimens from 9 patients with idiopathic adhesive capsulitis who had not shown improvement in symptoms after 5 months of physiotherapy. Adhesive capsulitis was confirmed in all patients by magnetic resonance imaging. We also obtained specimens from 8 control patients who had underwent surgery for acute acromioclavicular joint dislocation and who had radiological indication of glenohumeral capsule alteration based on arthroscopic evaluation. mRNA expression in the synovium/capsule specimens was analyzed by quantitative reverse transcription PCR. The B2M and HPRT1 genes were used as references to normalize target gene expression in the shoulder tissue samples. RESULTS: The synovium/capsule samples from the patients with adhesive capsulitis had significantly higher TNC and FN1 expression than those from the controls. Additionally, symptom duration directly correlated with expression of TGFβ1 receptor I. CONCLUSION: Elevated levels of TNC and FN1 expression may be a marker of capsule injury. Upregulation of TGFβ1 receptor I seems to be dependent on symptom duration; therefore, TGFβ signaling may be involved in adhesive capsulitis. As such, TNC, FN1 and TGFβ1 receptor I may also play roles in adhesive capsulitis by contributing to capsule inflammation and fibrosis.

  16. The roles of Tenascin C and Fibronectin 1 in adhesive capsulitis: a pilot gene expression study

    Science.gov (United States)

    Cohen, Carina; Leal, Mariana Ferreira; Belangero, Paulo Santoro; Figueiredo, Eduardo Antônio; Smith, Marília Cardoso; Andreoli, Carlos Vicente; de Castro Pochini, Alberto; Cohen, Moises; Ejnisman, Benno; Faloppa, Flávio

    2016-01-01

    OBJECTIVES: We evaluated mRNA expression levels of genes that encode TGF-β1; the TGF-β1 receptor; the collagen-modifying enzymes LOX, PLOD1, and PLOD2; and the extracellular matrix proteins COMP, FN1, TNC and TNXB in synovial/capsule specimens from patients with idiopathic adhesive capsulitis. Possible associations between the measured mRNA levels and clinical parameters were also investigated. METHODS: We obtained glenohumeral joint synovium/capsule specimens from 9 patients with idiopathic adhesive capsulitis who had not shown improvement in symptoms after 5 months of physiotherapy. Adhesive capsulitis was confirmed in all patients by magnetic resonance imaging. We also obtained specimens from 8 control patients who had underwent surgery for acute acromioclavicular joint dislocation and who had radiological indication of glenohumeral capsule alteration based on arthroscopic evaluation. mRNA expression in the synovium/capsule specimens was analyzed by quantitative reverse transcription PCR. The B2M and HPRT1 genes were used as references to normalize target gene expression in the shoulder tissue samples. RESULTS: The synovium/capsule samples from the patients with adhesive capsulitis had significantly higher TNC and FN1 expression than those from the controls. Additionally, symptom duration directly correlated with expression of TGFβ1 receptor I. CONCLUSION: Elevated levels of TNC and FN1 expression may be a marker of capsule injury. Upregulation of TGFβ1 receptor I seems to be dependent on symptom duration; therefore, TGFβ signaling may be involved in adhesive capsulitis. As such, TNC, FN1 and TGFβ1 receptor I may also play roles in adhesive capsulitis by contributing to capsule inflammation and fibrosis. PMID:27438566

  17. The flow of gene expression.

    Science.gov (United States)

    Misteli, Tom

    2004-03-01

    Gene expression is a highly interconnected multistep process. A recent meeting in Iguazu Falls, Argentina, highlighted the need to uncover both the molecular details of each single step as well as the mechanisms of coordination among processes in order to fully understand the expression of genes.

  18. Study of gene expression and OTA production by Penicillium nordicum during a small-scale seasoning process of salami.

    Science.gov (United States)

    Ferrara, Massimo; Magistà, Donato; Epifani, Filomena; Cervellieri, Salvatore; Lippolis, Vincenzo; Gallo, Antonia; Perrone, Giancarlo; Susca, Antonia

    2016-06-16

    Penicillium nordicum, an important and consistent producer of ochratoxin A (OTA), is a widely distributed contaminant of protein rich food with elevated NaCl. It is usually found on dry-cured meat products and is considered the main species responsible for their contamination by OTA. The aim of this work was to study the gene expression of a polyketide synthase (otapksPN) involved in P. nordicum OTA biosynthesis, and OTA production during a small-scale seasoning process. Fresh pork sausages were surface inoculated with P. nordicum and seasoned for 30days. Gene expression and OTA production were monitored throughout the seasoning process after 4, 5, 6, 7, 10, 14, and 30days. The expression of otapksPN gene was already detected after 4days and increased significantly after 7days of seasoning, reaching the maximum expression level after 10days (1.69×10(4)copies/100mg). Consistently with gene expression monitoring, OTA was detected from the 4th day and its content increased significantly from the 7th day, reaching the maximum level after 10days. In the late stages of the seasoning process, OTA did not increase further and the number of gene copies was progressively reduced after 14 and 30days.

  19. Microanalysis of gene expression in cultured cells

    NARCIS (Netherlands)

    E. van der Veer (Eveliene)

    1982-01-01

    textabstractIn this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing g

  20. Epstein-Barr virus-positive nodal peripheral T cell lymphomas: clinicopathologic and gene expression profiling study.

    Science.gov (United States)

    Ha, Sang Yun; Sung, Jiyeon; Ju, Hyunjung; Karube, Kennosuke; Kim, Soek Jin; Kim, Won Seog; Seto, Masao; Ko, Young-Hyeh

    2013-07-01

    Epstein-Barr virus-positive peripheral T cell lymphoma, not otherwise specified (EBV+ PTCL-NOS), in which virtually all neoplastic T cells harbor EBV, is a very rare disease with poor prognosis. To analyze the clinicopathologic characteristics and gene expression profile, we retrospectively collected six cases of EBV+ PTCL-NOS with no known primary immunodeficiency. The patients were 5 men and 1 woman, their age ranging from 48 years to 88 years (median 61.5 years). Lymphadenopathy was the most common presentation. Four patients had underlying disease, including HBV carrier, HCV infection, diabetes mellitus, and prostate cancer. All patients showed fatal clinical course in spite of chemotherapy. Histopathologically, monotonous infiltration of atypical lymphocytes of small to medium size was shown in four patients and medium to large tumor cells in two patients. Five patients showed CD4-/CD8+/bF-1+ phenotype with TIA-1 expression. In gene expression analysis using mRNA microarray, genes differentially expressed in EBV+ PTCL-NOS compared to normal reactive lymph nodes included 1515 genes (Mann-Whitney U-test pgenes involved in B cell differentiation or activation were mostly down-regulated, and T cell activation was mostly suppressed by down-regulation of activation genes and up-regulation of regulatory genes. Genes associated with cytotoxic activity were mostly up-regulated. Based on its peculiar clinical, histopathologic, and gene expression findings in EBV+ PTCL-NOS, we suggest EBV+ PTCL-NOS as a distinct disease entity from PTCL-NOS. In this study, the finding that most significantly enriched the functional term was immune response, suggesting a specific relation between EBV infection and alteration of immune response in the patients with EBV+ PTCL-NOS.

  1. Deriving Trading Rules Using Gene Expression Programming

    Directory of Open Access Journals (Sweden)

    Adrian VISOIU

    2011-01-01

    Full Text Available This paper presents how buy and sell trading rules are generated using gene expression programming with special setup. Market concepts are presented and market analysis is discussed with emphasis on technical analysis and quantitative methods. The use of genetic algorithms in deriving trading rules is presented. Gene expression programming is applied in a form where multiple types of operators and operands are used. This gives birth to multiple gene contexts and references between genes in order to keep the linear structure of the gene expression programming chromosome. The setup of multiple gene contexts is presented. The case study shows how to use the proposed gene setup to derive trading rules encoded by Boolean expressions, using a dataset with the reference exchange rates between the Euro and the Romanian leu. The conclusions highlight the positive results obtained in deriving useful trading rules.

  2. Time-course investigation of the gene expression profile during Fasciola hepatica infection: A microarray-based study

    Directory of Open Access Journals (Sweden)

    Jose Rojas-Caraballo

    2015-12-01

    Full Text Available Fasciolosis is listed as one of the most important neglected tropical diseases according with the World Health Organization and is also considered as a reemerging disease in the human beings. Despite there are several studies describing the immune response induced by Fasciola hepatica in the mammalian host, investigations aimed at identifying the expression profile of genes involved in inducing hepatic injury are currently scarce. Data presented here belong to a time-course investigation of the gene expression profile in the liver of BALB/c mice infected with F. hepatica metacercariae at 7 and 21 days after experimental infection. The data published here have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE69588, previously published by Rojas-Caraballo et al. (2015 in PLoS One [1].

  3. Studies of the Expression of Estrogen Receptor Gene in the Rat Uterus during the Estrous Cycle and Periimplantational Period

    Institute of Scientific and Technical Information of China (English)

    张沅; 秦武轩; 赵炳顺; 范植明; 邹继超; 张永莲

    1995-01-01

    The correlation of serum estradiol concentration and uterine estrogen receptor (ER) gene expression (ERn and ERe quantitated by Dextran Coat Charcoal assay and ER mRNA by Northern blotting) was studied during the rat estrous cycle and early pregnant stage (dl - d10). The ER gene expression was up-regulated by estrogen and the levels of ER mRNA synchronized with the changes of ER protein, suggesting that estrogen influenced the transcriptional step of the ER gene. Post-ceitum ER expression increased with the serum estradiol progressively, reached a peak on d4 -d5 (just before implantation), but drastically dropped to the nadir on d6-d7 (during implantation) and then recovered. It was of interest to discover that ER mRNA level in the nonhnplantation sites (NIS) of uterus was much higher than that in the implantation sites (IS).

  4. Cold Response of Dedifferentiated Barley Cells at the Gene Expression, Hormone Composition, and Freezing Tolerance Levels: Studies on Callus Cultures

    OpenAIRE

    Vashegyi, I.; Marozsan-Toth, Z.; Galiba, G.; Dobrev, P. (Petre); Vaňková, R. (Radomíra); Toth, B.

    2013-01-01

    In this study, data is presented how dark-grown, embryogenic barley callus cells respond to cold without any light-dependent, chloroplast-related mechanism, independently of the systemic signals. The expression of HvCBF9, HvCBF14, and HvCOR14b genes, members of one of the most important cold-inducible regulatory system, was measured by real-time PCR. Characteristic of the cold response was similar in the crowns of seedlings and in dark-grown callus cultures, however, gene expression levels we...

  5. A new experimental approach for studying bacterial genomic island evolution identifies island genes with bacterial host-specific expression patterns

    Directory of Open Access Journals (Sweden)

    Nickerson Cheryl A

    2006-01-01

    Full Text Available Abstract Background Genomic islands are regions of bacterial genomes that have been acquired by horizontal transfer and often contain blocks of genes that function together for specific processes. Recently, it has become clear that the impact of genomic islands on the evolution of different bacterial species is significant and represents a major force in establishing bacterial genomic variation. However, the study of genomic island evolution has been mostly performed at the sequence level using computer software or hybridization analysis to compare different bacterial genomic sequences. We describe here a novel experimental approach to study the evolution of species-specific bacterial genomic islands that identifies island genes that have evolved in such a way that they are differentially-expressed depending on the bacterial host background into which they are transferred. Results We demonstrate this approach by using a "test" genomic island that we have cloned from the Salmonella typhimurium genome (island 4305 and transferred to a range of Gram negative bacterial hosts of differing evolutionary relationships to S. typhimurium. Systematic analysis of the expression of the island genes in the different hosts compared to proper controls allowed identification of genes with genera-specific expression patterns. The data from the analysis can be arranged in a matrix to give an expression "array" of the island genes in the different bacterial backgrounds. A conserved 19-bp DNA site was found upstream of at least two of the differentially-expressed island genes. To our knowledge, this is the first systematic analysis of horizontally-transferred genomic island gene expression in a broad range of Gram negative hosts. We also present evidence in this study that the IS200 element found in island 4305 in S. typhimurium strain LT2 was inserted after the island had already been acquired by the S. typhimurium lineage and that this element is likely not

  6. Combination of cold atmospheric plasma and iron nanoparticles in breast cancer: gene expression and apoptosis study

    Directory of Open Access Journals (Sweden)

    Jalili A

    2016-09-01

    Full Text Available Azam Jalili,1 Shiva Irani,1 Reza Mirfakhraie2 1Department of Biology, Science and Research Branch, Islamic Azad University, 2Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran Background: Current cancer treatments have unexpected side effects of which the death of normal cells is one. In some cancers, iron nanoparticles (NPs can be subjected to diagnosis and passive targeting treatment. Cold atmospheric plasma (CAP has a proven induction of selective cell death ability. In this study, we have attempted to analyze the synergy between CAP and iron NPs in human breast adenocarcinoma cells (MCF-7.Materials and methods: In vitro cytotoxicity of CAP treatment and NPs in cells measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and cell death was shown by 4',6-diamidino-2-phenylindole and annexin V staining. Fluctuations in BAX and BCL-2 gene expression were investigated by means of real-time polymerase chain reaction.Results: MTT assay results showed that combination of plasma and iron NPs decreased the viability of cancer cells significantly (P<0.05. Real-time analysis showed that the combination therapy induced shifting the BAX/BCL-2 ratio in favor of apoptosis.Conclusion: Our data indicate that synergy between CAP and iron NPs can be applied in breast cancer treatment selectively. Keywords: breast cancer, cold atmospheric plasma, iron nanoparticles, BAX, BCL-2

  7. An optimised direct lysis method for gene expression studies on low cell numbers.

    Science.gov (United States)

    Le, Anh Viet-Phuong; Huang, Dexing; Blick, Tony; Thompson, Erik W; Dobrovic, Alexander

    2015-08-05

    There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.

  8. Gene expression profiling during murine tooth development

    Directory of Open Access Journals (Sweden)

    Maria A dos Santos silva Landin

    2012-07-01

    Full Text Available The aim of this study was to describe the expression of genes, including ameloblastin (Ambn, amelogenin X chromosome (Amelx and enamelin (Enam during early (pre-secretory tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24h intervals, starting at the eleventh embryonic day (E11.5 and up to the seventh day after birth (P7. The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx and Enam. Microarray results where validated using real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR, and translated proteins identified by Western blotting. In situ localisation of the Ambn, Amelx and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially (p ≤0.05 expressed (DE genes.Microarray results showed a total of 4362 genes including Ambn, Amelx and Enam to be significant differentially expressed throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5-P0 increasing after birth (P1-P7. Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. The mRNAs expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around thirty-five genes were associated with fifteen transcription factors.

  9. Gene Expression Profiling of Gastric Cancer

    Science.gov (United States)

    Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

    2015-01-01

    Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

  10. Development of a RNA extraction method from milk for gene expression study in the mammary gland of sheep.

    Science.gov (United States)

    Mura, Maria Consuelo; Daga, Cinzia; Bodano, Sara; Paludo, Marta; Luridiana, Sebastiano; Pazzola, Michele; Dettori, Maria Luisa; Vacca, Giuseppe Massimo; Carcangiu, Vincenzo

    2013-03-01

    The aim of the study was to develop a reliable method for the RNA extraction from milk of Sarda sheep breed and to highlight if the extracted RNA can be used for expression study on mammary genes involved in milk fat synthesis using RT-qPCR. The main result is that a sample of 150 ml of milk provides an optimal amount of RNA (73.5 μg/ml). The highest RNA concentration has been found in the samples analysed within 4 h after collection. The RNA extracted was positively correlated to the number of somatic cells (P Ct value, for SREBPF1 gene of 26.8 ± 0.15. This research demonstrated that the high-quality of the RNA obtained is suited to use for studies of mammary genes expression in sheep, avoiding any damage caused by mammary gland biopsy.

  11. Gene set analysis for longitudinal gene expression data

    Directory of Open Access Journals (Sweden)

    Piepho Hans-Peter

    2011-07-01

    Full Text Available Abstract Background Gene set analysis (GSA has become a successful tool to interpret gene expression profiles in terms of biological functions, molecular pathways, or genomic locations. GSA performs statistical tests for independent microarray samples at the level of gene sets rather than individual genes. Nowadays, an increasing number of microarray studies are conducted to explore the dynamic changes of gene expression in a variety of species and biological scenarios. In these longitudinal studies, gene expression is repeatedly measured over time such that a GSA needs to take into account the within-gene correlations in addition to possible between-gene correlations. Results We provide a robust nonparametric approach to compare the expressions of longitudinally measured sets of genes under multiple treatments or experimental conditions. The limiting distributions of our statistics are derived when the number of genes goes to infinity while the number of replications can be small. When the number of genes in a gene set is small, we recommend permutation tests based on our nonparametric test statistics to achieve reliable type I error and better power while incorporating unknown correlations between and within-genes. Simulation results demonstrate that the proposed method has a greater power than other methods for various data distributions and heteroscedastic correlation structures. This method was used for an IL-2 stimulation study and significantly altered gene sets were identified. Conclusions The simulation study and the real data application showed that the proposed gene set analysis provides a promising tool for longitudinal microarray analysis. R scripts for simulating longitudinal data and calculating the nonparametric statistics are posted on the North Dakota INBRE website http://ndinbre.org/programs/bioinformatics.php. Raw microarray data is available in Gene Expression Omnibus (National Center for Biotechnology Information with

  12. Validation of reference genes for gene expression studies in the dinoflagellateAkashiwo sanguinea by quantitative real-time RT-PCR

    Institute of Scientific and Technical Information of China (English)

    DENG Yunyan; HU Zhangxi; MA Zhaopeng; TANG Ying Zhong

    2016-01-01

    The accurate measurement of gene expression via quantitative real-time reverse transcription PCR (qRT-PCR) heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of qRT-PCR to the HAB-forming group. In this study, six candidate reference genes, 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH),α-tubulin (TUA),β-tubulin (TUB), actin (ACT) and cytochrome oxidase subunit 1 (COX1), were evaluated for their expression stability with qRT-PCR and three statistical algorithms (GeNorm, NormFinder, and BestKeeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellateAkashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages (resting cysts), different growth stages, in darkness, exposed to abscisic acid (ABA) and exposed to temperature stress. The results indicated thatTUA,18S andGAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs ofACT andTUA,18S andGAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination ofTUA andTUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair ofACT andCOX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle, particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates

  13. Altered Cytokine Gene Expression in Peripheral Blood Monocytes across the Menstrual Cycle in Primary Dysmenorrhea: A Case-Control Study

    Science.gov (United States)

    Ma, Hongyue; Hong, Min; Duan, Jinao; Liu, Pei; Fan, Xinsheng; Shang, Erxin; Su, Shulan; Guo, Jianming; Qian, Dawei; Tang, Yuping

    2013-01-01

    Primary dysmenorrhea is one of the most common gynecological complaints in young women, but potential peripheral immunologic features underlying this condition remain undefined. In this paper, we compared 84 common cytokine gene expression profiles of peripheral blood mononuclear cells (PBMCs) from six primary dysmenorrheic young women and three unaffected controls on the seventh day before (secretory phase), and the first (menstrual phase) and the fifth (regenerative phase) days of menstruation, using a real-time PCR array assay combined with pattern recognition and gene function annotation methods. Comparisons between dysmenorrhea and normal control groups identified 11 (nine increased and two decreased), 14 (five increased and nine decreased), and 15 (seven increased and eight decreased) genes with ≥2-fold difference in expression (Pmenstrual phase, genes encoding pro-inflammatory cytokines (IL1B, TNF, IL6, and IL8) were up-regulated, and genes encoding TGF-β superfamily members (BMP4, BMP6, GDF5, GDF11, LEFTY2, NODAL, and MSTN) were down-regulated. Functional annotation revealed an excessive inflammatory response and insufficient TGF-β superfamily member signals with anti-inflammatory consequences, which may directly contribute to menstrual pain. In the secretory and regenerative phases, increased expression of pro-inflammatory cytokines and decreased expression of growth factors were also observed. These factors may be involved in the regulation of decidualization, endometrium breakdown and repair, and indirectly exacerbate primary dysmenorrhea. This first study of cytokine gene expression profiles in PBMCs from young primary dysmenorrheic women demonstrates a shift in the balance between expression patterns of pro-inflammatory cytokines and TGF-β superfamily members across the whole menstrual cycle, underlying the peripheral immunologic features of primary dysmenorrhea. PMID:23390521

  14. Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Longjian Niu

    2015-06-01

    Full Text Available Real-time quantitative PCR (RT-qPCR is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis, a promising oilseed crop known for its polyunsaturated fatty acid (PUFA-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt, BestKeeper, geNorm, and NormFinder were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE, actin (ACT and phospholipase A22 (PLA were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13, cyclophilin (CYC and elongation factor-1alpha (EF1α were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

  15. The Beta-Binomial Distribution for Estimating the Number of False Rejections in Microarray Gene Expression Studies.

    Science.gov (United States)

    Hunt, Daniel L; Cheng, Cheng; Pounds, Stanley

    2009-03-15

    In differential expression analysis of microarray data, it is common to assume independence among null hypotheses (and thus gene expression levels). The independence assumption implies that the number of false rejections V follows a binomial distribution and leads to an estimator of the empirical false discovery rate (eFDR). The number of false rejections V is modeled with the beta-binomial distribution. An estimator of the beta-binomial false discovery rate (bbFDR) is then derived. This approach accounts for how the correlation among non-differentially expressed genes influences the distribution of V. Permutations are used to generate the observed values for V under the null hypotheses and a beta-binomial distribution is fit to the values of V. The bbFDR estimator is compared to the eFDR estimator in simulation studies of correlated non-differentially expressed genes and is found to outperform the eFDR for certain scenarios. As an example, this method is also used to perform an analysis that compares the gene expression of soft tissue sarcoma samples to normal tissue samples.

  16. Prophylactic effect of Mucuna pruriens Linn (velvet bean) seed extract against experimental Naja sputatrix envenomation: gene expression studies.

    Science.gov (United States)

    Fung, Shin Yee; Sim, Si Mui; Kandiah; Jeyaseelan; Armugam, Arunmozhiarasi; Aguiyi, John Chinyere; Tan, Nget Hong

    2014-09-01

    Mucuna pruriens is widely used in traditional medicine for treatments of various diseases. In certain region of Nigeria, the seed is used as oral prophylactics for snakebite. Rats pretreated with the aqueous extract from M. pruriens seed (MPE) were protected against the lethal effects of Naja sputatrix (Javan spitting cobra) venom [Tan et al., J Ethnopharmacol, 123 (2009) 356]. The pretreatment also protected against venom-induced histopathological changes in rat heart. To contribute to the understanding of the mechanism of cardio-protective action, the present study examined the effects of MPE-pretreatment on gene expression profile of rat heart as well as effect of MPE-pretreatment on N. sputatrix venom-induced gene expression alterations in rat heart. The gene expression profiles were examined by microarray analysis and verified by real time PCR. The results showed that pretreatment with MPE caused 50 genes in the rat heart substantially up-regulated of which 19 were related to immune responses, 7 were related to energy production and metabolism. The up-regulation of genes related to energy metabolism probably plays a role in maintaining the viability of the heart. Four other genes that were up-regulated (alpha synuclein, natriuretic peptide precursor, calsequestrin and triadin) were involved in the maintenance of homeostasis of the heart or maintaining its viability, thereby contributing to the direct protective action. The results demonstrated that protective effect of MPE pretreatment against snake venom poisoning may involve a direct action on the heart.

  17. Foxp3 gene expression in oral lichen planus: A clinicopathological study

    National Research Council Canada - National Science Library

    LEI, LEI; ZHAN, LIHUA; TAN, WEIXIA; CHEN, SHAOHUA; LI, YANGQIU; REYNOLDS, MARK

    2014-01-01

    ...). The present study aimed to investigate Foxp3 expression in CD4+CD25+ T cells of peripheral blood mononuclear cells and oral lesions in patients diagnosed with OLP, who were grouped as OLP subtype, duration and relapse...

  18. [Study on the expressions of basic fibroblast growth factor and nervous growth factor genes in rat cerebral concussion].

    Science.gov (United States)

    Peng, Rui-yun; Gao, Ya-bing; Xiao, Xing-yi; Wang, De-wen; Chen, Hao-yu; Wu, Xiao-hong; Liu, Jie; Hu, Wen-hua; Cai, Bao-ren

    2003-04-01

    To study the expressions of basic fibroblast growth factor (bFGF) and nervous growth factor(NGF) genes in rat cerebral concussion. Eighty Wistar male rats were used for animal model of cerebral concussion, which were sacrificed on 1, 3, 7, 14 and 30 days after injury and the brain tissue was taken out. The expressions of bFGF and NGF genes were studied in the course of cerebral concussion by means of immunohistochemistry and in situ hybridization. Rats in 100 g group were seen the clinical manifestation for typical cerebral concussion. The protein and mRNA of bFGF were increased on day 1, obtained at peak on day 3-7, decreased on day 14 and also increased on day 30 compared with controls. The positive area was seen in the plasma of neurons in cerebral cortex, hippocampus, thalamus and cerebellum. NGF protein and mRNA showed strong positive and increased in the plasma of neurons in cerebral cortex, hippocampus, thalamus and cerebellum on day 1, and they were continuously positive but gradually decreased within 30 days after injury. The expression of bFGF gene participates in the course of cerebral concussion, might play an important role in the nervous cells degeneration and necrosis; NGF gene expression participates in the whole course of cerebral concussion, especially in the early phase.

  19. Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR.

    Science.gov (United States)

    Du, Yishuai; Zhang, Linlin; Xu, Fei; Huang, Baoyu; Zhang, Guofan; Li, Li

    2013-03-01

    Hatchery-reared larvae of the Pacific oyster (Crassostrea gigas) often suffer from massive mortality induced by Ostreid herpesvirus 1 (OsHV-1) infection, indicating the importance of better understanding of oyster immune defense systems. The accuracy of measurements of gene expression levels based on quantitative real-time PCR assays relies on the use of housekeeping genes as internal controls; however, few studies have focused on the selection of such internal controls. In this study, we conducted a comprehensive investigation of internal control genes during oyster development in virus-infected and uninfected samples. Transcriptome data for 38 developmental stages were downloaded and the gene expression patterns were classified into 30 clusters. A total of 317 orthologs of classical housekeeping genes in the oyster genome were annotated. After combining the expression profiles and oyster housekeeping gene dataset, 14 candidate internal controls were selected for further investigation: Elongation factor-1α (EF-1α), 18S rRNA (18S), 28S rRNA (28S), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin (ACT), Ribosomal protein L7 (RL7), Ribosomal protein L27 (RL27), Ribosomal protein L36 (RL36), Ribosomal protein S18 (RS18), Heterogeneous nuclear ribonucleoprotein A2/B1 (RO21), Eukaryotic translation elongation factor 2 (EF2), Ubiquitin-conjugating enzyme E2D2 (UBCD1), S-phase kinase-associated protein 1 (SKP1) and Heterogeneous nuclear ribonucleoprotein Q (HNRPQ). RNA was extracted from oyster larvae infected with OsHV-1 (group A; GA), and OsHV-1 free larvae (group B; GB). The expression levels of the 14 candidate internal controls were studied in GA and GB larvae by real-time PCR. Their expression stabilities were further analyzed using the GeNorm program. RL7 and RS18 were the most stable genes in both OsHV-1 infected (GA) and uninfected (GB) larvae. These results suggest that RL7 and RS18 could be used as internal controls for studying gene expression in

  20. Correlative study of peripheral ATP1A1 gene expression level to anxiety severity score on major depressive disorder patients.

    Science.gov (United States)

    Zhao, Jingjie; Guo, Xu; Du, Yi; Han, Yu; Wang, Yongzhi; Li, Li; Qian, Jialin; Li, Mingzhen; Wu, Huijuan; Golden, Teresa; Wu, Ning

    2016-11-01

    Major depressive disorder (MDD) frequently co-occurs with other psychiatric problems. Our previous study showed that ATP1A1 gene expression level was significantly decreased in MDD patients. This research explores the potential correlations between the ATP1A1 expression level reduction and MDD patients' clinical manifestation. All participant patients were diagnosed by Diagnostic and Statistical Manual of Mental Disorders - 4th edition (DSM-IV). Hamilton rating scale for depression (HAM-D) and anxiety (HAM-A) were applied to group patients into different categories. ATP1A1 expression level was measured by reverse transcript real-time polymerase chain reaction. ATP1A1 expression levels of all MDD subgroups showed significant reduction compared to the control group (p0.05). ATP1A1 expression level reduction is related to MDD anxiety score, which may be an explanation for the clinical manifestations and the underlining physiological mechanisms.

  1. A growth curve model with fractional polynomials for analysing incomplete time-course data in microarray gene expression studies

    DEFF Research Database (Denmark)

    Tan, Qihua; Thomassen, Mads; Hjelmborg, Jacob V B

    2011-01-01

    among fractional polynomial models with power terms from a set of fixed values that offer a wide range of curve shapes and suggests a best fitting model. After a limited simulation study, the model has been applied to our human in vivo irritated epidermis data with missing observations to investigate......-course pattern in a gene by gene manner. We introduce a growth curve model with fractional polynomials to automatically capture the various time-dependent expression patterns and meanwhile efficiently handle missing values due to incomplete observations. For each gene, our procedure compares the performances...... time-dependent transcriptional responses to a chemical irritant. Our method was able to identify the various nonlinear time-course expression trajectories. The integration of growth curves with fractional polynomials provides a flexible way to model different time-course patterns together with model...

  2. Identification and evaluation of suitable reference genes for gene expression studies in the whitefly Bemisia tabaci (Asia I) by reverse transcription quantitative realtime PCR.

    Science.gov (United States)

    Collins, Carl; Patel, Mitulkumar V; Colvin, John; Bailey, David; Seal, Susan

    2014-05-02

    This study presents a reliable method for performing reverse transcription quantitative realtime PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data pre- sented here will assist future transcript profiling studies in whiteflies.

  3. Identification and Evaluation of Suitable Reference Genes for Gene Expression Studies in the Whitefly Bemisia tabaci (Asia I) by Reverse Transcription Quantitative Real-Time PCR

    Science.gov (United States)

    Collins, Carl; Patel, Mitulkumar V.; Colvin, John; Bailey, David; Seal, Susan

    2014-01-01

    This study presents a reliable method for performing reverse transcription quantitative real-time PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data presented here will assist future transcript profiling studies in whiteflies. PMID:25373210

  4. Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study

    Directory of Open Access Journals (Sweden)

    Akahoshi Eiichi

    2009-06-01

    Full Text Available Abstract Background Dioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR. This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR. Methods N2a-Rβ cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA. Results Induction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE, though AHRE-III was not identical to XRE, the conventional AhR-binding motif. Conclusion Our results suggest TCDD directly regulate the dopamine system by TH gene

  5. Lean mass, muscle strength and gene expression in community dwelling older men: findings from the Hertfordshire Sarcopenia Study (HSS).

    Science.gov (United States)

    Patel, Harnish P; Al-Shanti, Nasser; Davies, Lucy C; Barton, Sheila J; Grounds, Miranda D; Tellam, Ross L; Stewart, Claire E; Cooper, Cyrus; Sayer, Avan Aihie

    2014-10-01

    Sarcopenia is associated with adverse health outcomes. This study investigated whether skeletal muscle gene expression was associated with lean mass and grip strength in community-dwelling older men. Utilising a cross-sectional study design, lean muscle mass and grip strength were measured in 88 men aged 68-76 years. Expression profiles of 44 genes implicated in the cellular regulation of skeletal muscle were determined. Serum was analysed for circulating cytokines TNF (tumour necrosis factor), IL-6 (interleukin 6, IFNG (interferon gamma), IL1R1 (interleukin-1 receptor-1). Relationships between skeletal muscle gene expression, circulating cytokines, lean mass and grip strength were examined. Participant groups with higher and lower values of lean muscle mass (n = 18) and strength (n = 20) were used in the analysis of gene expression fold change. Expression of VDR (vitamin D receptor) [fold change (FC) 0.52, standard error for fold change (SE) ± 0.08, p = 0.01] and IFNG mRNA (FC 0.31; SE ± 0.19, p = 0.01) were lower in those with higher lean mass. Expression of IL-6 (FC 0.43; SE ± 0.13, p = 0.02), TNF (FC 0.52; SE ± 0.10, p = 0.02), IL1R1 (FC 0.63; SE ± 0.09, p = 0.04) and MSTN (myostatin) (FC 0.64; SE ± 0.11, p = 0.04) were lower in those with higher grip strength. No other significant changes were observed. Significant negative correlations between serum IL-6 (R = -0.29, p = 0.005), TNF (R = -0.24, p = 0.017) and grip strength were demonstrated. This novel skeletal muscle gene expression study carried out within a well-characterized epidemiological birth cohort has demonstrated that lower expression of VDR and IFNG is associated with higher lean mass, and lower expression of IL-6, TNF, IL1R1 and myostatin is associated with higher grip strength. These findings are consistent with a role of proinflammatory factors in mediating lower muscle strength in community-dwelling older men.

  6. Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives

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    Hummel Oliver

    2009-02-01

    Full Text Available Abstract Background Mouse embryonic stem (ES cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF. Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. Results By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. Conclusion We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment, which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG is still expressed and which might be down

  7. Comprehensive selection of reference genes for expression studies in meniscus injury using quantitative real-time PCR.

    Science.gov (United States)

    Leal, Mariana Ferreira; Arliani, Gustavo Gonçalves; Astur, Diego Costa; Franciozi, Carlos Eduardo; Debieux, Pedro; Andreoli, Carlos Vicente; Smith, Marília Cardoso; Pochini, Alberto de Castro; Ejnisman, Benno; Cohen, Moises

    2016-06-10

    The meniscus plays critical roles in the knee function. Meniscal tears can lead to knee osteoarthritis. Gene expression analysis may be a useful tool for understanding meniscus tears, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. We evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using meniscus samples of (1) 19 patients with isolated meniscal tears, (2) 20 patients with meniscal tears and combined anterior cruciate ligament injury (ACL), and (3) 11 controls without meniscal tears. The stability of the candidate reference genes was determined using the NormFinder, geNorm, BestKeeper DataAssist and RefFinder software packages and comparative ΔCt method. Overall, HPRT1 was the best single reference gene. However, GenEx software demonstrated that two or more reference genes should be used for gene expression normalization, which was confirmed when we evaluated TGFβR1 expression using several reference gene combinations. HPRT1+TBP was the most frequently identified pair from the analysis of samples of (1) meniscal tear samples of patients with a concomitant ACL tears, (2) all meniscal tears, and (3) all samples. HPRT1+GAPDH was the most frequently identified pair from the analysis of samples of isolated meniscal tear samples and controls. In the analysis involving only controls, GAPDH+18S was the most frequently identified pair. In the analysis of only isolated meniscal tear samples and in the analysis of meniscal tear samples of patients with concomitant ACL tears and controls, both HPRT1+TBP and HPRT1+GAPDH were identified as suitable pairs. If the gene expression study aims to compare non-injured meniscus, isolated meniscal tears and meniscal tears of patients with ACL tears as three independent groups, the trio of HPRT1+TBP+GAPDH is the most suitable

  8. Cloning of BMP-2 Gene and Its Expression Study in E. coli in Order to Produce a Recombinant Drug

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    N. Mohammadi

    2014-10-01

    Full Text Available Introduction & Objective: Bone morphogenetic proteins are a group of cytokines that belongs to superfamily TGF?. These proteins play an important role in evolution of many of organs and tissues through germinal period followed by amending and rebuilding of bone tissue and car-tilage. The aim of this study was to clone and expression analysis of BMP-2 gene in E. coli bacteria. Materials & Methods: In this experimental study the sequence of cDNA related to the mature peptide of human morphogenetic protein-2 (BMP-2 in E.coli was synthesized and cloned in a PET system. After sequencing, recombinant plasmid pET28a/BMP-2 was transformed into the expression host, E.coli BL21 (DE3. The transformed bacteria were cultured in LB me-dium containing kanamaycin antibiotic at 37° C for O/N. Then, induction with IPTG took place. The expression was evaluated by reverse transcriptase PCR and SDS-PAGE followed by western blotting to confirm its identity. The observed band on SDS-PSGE showed the presence of the expressed protein at the 14k Dalton segment which was confirmed by west-ern blotting technique. Results: The gene sequence was amplified by PCR. After gene and plasmid preparation, lega-tion was performed. Sequencing confirmed accuracy of cloning. Protein expression was demonstrated by RT-PCR and SDS-PAGE. Results were confirmed by western blotting. Conclusion: In this study over-expression of this recombinant protein was achieved in a pro-karyotic system. Different concentrations of inducer were applied and harvesting was per-formed in different times after induction. The best expression was detected in 4 hours after induction with a concentration of 1mM IPTG. (Sci J Hamadan Univ Med Sci 2014; 21 (3: 196-202

  9. Partial Sleep Restriction Activates Immune Response-Related Gene Expression Pathways: Experimental and Epidemiological Studies in Humans

    Science.gov (United States)

    Rantanen, Ville; Kronholm, Erkki; Surakka, Ida; van Leeuwen, Wessel M. A.; Lehto, Maili; Matikainen, Sampsa; Ripatti, Samuli; Härmä, Mikko; Sallinen, Mikael; Salomaa, Veikko; Jauhiainen, Matti; Alenius, Harri; Paunio, Tiina; Porkka-Heiskanen, Tarja

    2013-01-01

    Epidemiological studies have shown that short or insufficient sleep is associated with increased risk for metabolic diseases and mortality. To elucidate mechanisms behind this connection, we aimed to identify genes and pathways affected by experimentally induced, partial sleep restriction and to verify their connection to insufficient sleep at population level. The experimental design simulated sleep restriction during a working week: sleep of healthy men (N = 9) was restricted to 4 h/night for five nights. The control subjects (N = 4) spent 8 h/night in bed. Leukocyte RNA expression was analyzed at baseline, after sleep restriction, and after recovery using whole genome microarrays complemented with pathway and transcription factor analysis. Expression levels of the ten most up-regulated and ten most down-regulated transcripts were correlated with subjective assessment of insufficient sleep in a population cohort (N = 472). Experimental sleep restriction altered the expression of 117 genes. Eight of the 25 most up-regulated transcripts were related to immune function. Accordingly, fifteen of the 25 most up-regulated Gene Ontology pathways were also related to immune function, including those for B cell activation, interleukin 8 production, and NF-κB signaling (P<0.005). Of the ten most up-regulated genes, expression of STX16 correlated negatively with self-reported insufficient sleep in a population sample, while three other genes showed tendency for positive correlation. Of the ten most down-regulated genes, TBX21 and LGR6 correlated negatively and TGFBR3 positively with insufficient sleep. Partial sleep restriction affects the regulation of signaling pathways related to the immune system. Some of these changes appear to be long-lasting and may at least partly explain how prolonged sleep restriction can contribute to inflammation-associated pathological states, such as cardiometabolic diseases. PMID:24194869

  10. Partial sleep restriction activates immune response-related gene expression pathways: experimental and epidemiological studies in humans.

    Directory of Open Access Journals (Sweden)

    Vilma Aho

    Full Text Available Epidemiological studies have shown that short or insufficient sleep is associated with increased risk for metabolic diseases and mortality. To elucidate mechanisms behind this connection, we aimed to identify genes and pathways affected by experimentally induced, partial sleep restriction and to verify their connection to insufficient sleep at population level. The experimental design simulated sleep restriction during a working week: sleep of healthy men (N = 9 was restricted to 4 h/night for five nights. The control subjects (N = 4 spent 8 h/night in bed. Leukocyte RNA expression was analyzed at baseline, after sleep restriction, and after recovery using whole genome microarrays complemented with pathway and transcription factor analysis. Expression levels of the ten most up-regulated and ten most down-regulated transcripts were correlated with subjective assessment of insufficient sleep in a population cohort (N = 472. Experimental sleep restriction altered the expression of 117 genes. Eight of the 25 most up-regulated transcripts were related to immune function. Accordingly, fifteen of the 25 most up-regulated Gene Ontology pathways were also related to immune function, including those for B cell activation, interleukin 8 production, and NF-κB signaling (P<0.005. Of the ten most up-regulated genes, expression of STX16 correlated negatively with self-reported insufficient sleep in a population sample, while three other genes showed tendency for positive correlation. Of the ten most down-regulated genes, TBX21 and LGR6 correlated negatively and TGFBR3 positively with insufficient sleep. Partial sleep restriction affects the regulation of signaling pathways related to the immune system. Some of these changes appear to be long-lasting and may at least partly explain how prolonged sleep restriction can contribute to inflammation-associated pathological states, such as cardiometabolic diseases.

  11. Preprocessing and Quality Control Strategies for Illumina DASL Assay-Based Brain Gene Expression Studies with Semi-Degraded Samples.

    Science.gov (United States)

    Chow, Maggie L; Winn, Mary E; Li, Hai-Ri; April, Craig; Wynshaw-Boris, Anthony; Fan, Jian-Bing; Fu, Xiang-Dong; Courchesne, Eric; Schork, Nicholas J

    2012-01-01

    Available statistical preprocessing or quality control analysis tools for gene expression microarray datasets are known to greatly affect downstream data analysis, especially when degraded samples, unique tissue samples, or novel expression assays are used. It is therefore important to assess the validity and impact of the assumptions built in to preprocessing schemes for a dataset. We developed and assessed a data preprocessing strategy for use with the Illumina DASL-based gene expression assay with partially degraded postmortem prefrontal cortex samples. The samples were obtained from individuals with autism as part of an investigation of the pathogenic factors contributing to autism. Using statistical analysis methods and metrics such as those associated with multivariate distance matrix regression and mean inter-array correlation, we developed a DASL-based assay gene expression preprocessing pipeline to accommodate and detect problems with microarray-based gene expression values obtained with degraded brain samples. Key steps in the pipeline included outlier exclusion, data transformation and normalization, and batch effect and covariate corrections. Our goal was to produce a clean dataset for subsequent downstream differential expression analysis. We ultimately settled on available transformation and normalization algorithms in the R/Bioconductor package lumi based on an assessment of their use in various combinations. A log2-transformed, quantile-normalized, and batch and seizure-corrected procedure was likely the most appropriate for our data. We empirically tested different components of our proposed preprocessing strategy and believe that our results suggest that a preprocessing strategy that effectively identifies outliers, normalizes the data, and corrects for batch effects can be applied to all studies, even those pursued with degraded samples.

  12. Intra-city Differences in Cardiac Expression of Inflammatory Genes and Inflammasomes in Young Urbanites: A Pilot Study

    OpenAIRE

    Villarreal-Calderon, Rodolfo; Dale, Gary; Delgado-Chávez, Ricardo; Torres-Jardón, Ricardo; Zhu, Hongtu; Herritt, Lou; Gónzalez-Maciel, Angelica; Reynoso-Robles, Rafael; Yuan, Ying; Wang, Jiaping; Solorio-López, Edelmira; Medina-Cortina, Humberto; Calderón-Garcidueñas, Lilian

    2012-01-01

    Southwest Mexico City (SWMC) air pollution is characterized by high concentrations of ozone and particulate matter < 10 μm (PM10) containing lipopolysaccharides while in the North PM2.5 is high. These intra-city differences are likely accounting for higher CD14 and IL-1β in SWMC v NMC mice myocardial expression. This pilot study was designed to investigate whether similar intra-city differences exist in the levels of myocardial inflammatory genes in young people. Inflammatory mediator genes a...

  13. A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

    Science.gov (United States)

    Xie, Jinger; Nair, Ayyappan; Hermiston, Terry W

    2008-02-01

    Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.

  14. Studies on Expression of IGF-II Gene in Deciduas Derived from Medical Abortion Patients

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To determine the effect of insulin-like growth factor-Ⅱ (IGF-Ⅱ ) upon the maintenance of decidua in early pregnancy and its relationship with progesterone, as well as its role in medical abortion. Materials & Methods Decidua tissue was obtained from 28 women who undergoing surgical abortion and 39 for medical abortion respectively at 5~7 weeks of gestation. The extracted total RNA was reversely transcripted and amplified by PCR with spe cific primers (IGF-Ⅱ and β-actin). The products were semi-quantitated by MIAS 300 system and qualitatively analyzed by southern blotting. Results The expression of IGF-Ⅱ gene in decidua from surgical abortion was signif icantly higher than that from medical abortion (P<0.05). The average IGF-Ⅱ gene transcription values were 1. 54±0.79 and 0.72±0.39 respectively. The results of southern blotting proved qualitatively that the RT-PCR products were IGF-Ⅱ cDNA. Conclusion IGF-Ⅱ plays a role in the maintenance of decidua in early pregnancy. It may act as a mediator of progestin. It's also involved in the molecular mechanism of mifepristone.

  15. Integrating genome-wide association study and expression quantitative trait loci data identifies multiple genes and gene set associated with neuroticism.

    Science.gov (United States)

    Fan, Qianrui; Wang, Wenyu; Hao, Jingcan; He, Awen; Wen, Yan; Guo, Xiong; Wu, Cuiyan; Ning, Yujie; Wang, Xi; Wang, Sen; Zhang, Feng

    2017-08-01

    Neuroticism is a fundamental personality trait with significant genetic determinant. To identify novel susceptibility genes for neuroticism, we conducted an integrative analysis of genomic and transcriptomic data of genome wide association study (GWAS) and expression quantitative trait locus (eQTL) study. GWAS summary data was driven from published studies of neuroticism, totally involving 170,906 subjects. eQTL dataset containing 927,753 eQTLs were obtained from an eQTL meta-analysis of 5311 samples. Integrative analysis of GWAS and eQTL data was conducted by summary data-based Mendelian randomization (SMR) analysis software. To identify neuroticism associated gene sets, the SMR analysis results were further subjected to gene set enrichment analysis (GSEA). The gene set annotation dataset (containing 13,311 annotated gene sets) of GSEA Molecular Signatures Database was used. SMR single gene analysis identified 6 significant genes for neuroticism, including MSRA (p value=2.27×10(-10)), MGC57346 (p value=6.92×10(-7)), BLK (p value=1.01×10(-6)), XKR6 (p value=1.11×10(-6)), C17ORF69 (p value=1.12×10(-6)) and KIAA1267 (p value=4.00×10(-6)). Gene set enrichment analysis observed significant association for Chr8p23 gene set (false discovery rate=0.033). Our results provide novel clues for the genetic mechanism studies of neuroticism. Copyright © 2017. Published by Elsevier Inc.

  16. Regulation of meiotic gene expression in plants

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    Adele eZhou

    2014-08-01

    Full Text Available With the recent advances in genomics and sequencing technologies, databases of transcriptomes representing many cellular processes have been built. Meiotic transcriptomes in plants have been studied in Arabidopsis thaliana, rice (Oryza sativa, wheat (Triticum aestivum, petunia (Petunia hybrida, sunflower (Helianthus annuus, and maize (Zea mays. Studies in all organisms, but particularly in plants, indicate that a very large number of genes are expressed during meiosis, though relatively few of them seem to be required for the completion of meiosis. In this review, we focus on gene expression at the RNA level and analyze the meiotic transcriptome datasets and explore expression patterns of known meiotic genes to elucidate how gene expression could be regulated during meiosis. We also discuss mechanisms, such as chromatin organization and non-coding RNAs, that might be involved in the regulation of meiotic transcription patterns.

  17. Orally administered lactoperoxidase increases expression of the FK506 binding protein 5 gene in epithelial cells of the small intestine of mice: a DNA microarray study.

    Science.gov (United States)

    Wakabayashi, Hiroyuki; Miyauchi, Hirofumi; Shin, Kouichirou; Yamauchi, Koji; Matsumoto, Ichiro; Abe, Keiko; Takase, Mitsunori

    2007-09-01

    Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.

  18. Fruit and vegetable consumption and proinflammatory gene expression from peripheral blood mononuclear cells in young adults: a translational study

    Directory of Open Access Journals (Sweden)

    Puchau Blanca

    2010-05-01

    Full Text Available Abstract Background Fruits and vegetables are important sources of fiber and nutrients with a recognized antioxidant capacity, which could have beneficial effects on the proinflammatory status as well as some metabolic syndrome and cardiovascular disease features. The current study assessed the potential relationships of fruit and vegetable consumption with the plasma concentrations and mRNA expression values of some proinflammatory markers in young adults. Methods One-hundred and twenty healthy subjects (50 men/70 women; 20.8 ± 2.6 y; 22.3 ± 2.8 kg/m2 were enrolled. Experimental determinations included anthropometry, blood pressure and lifestyle features as well as blood biochemical and inflammatory measurements. The mRNA was isolated from peripheral blood mononuclear cells (PBMC and the gene expression concerning selected inflammatory markers was assessed by quantitative real-time PCR. Nutritional intakes were estimated by a validated semi-quantitative food-frequency questionnaire. Results The highest tertile of energy-adjusted fruit and vegetable consumption (>660 g/d was associated with lower plasma concentrations of C-reactive protein (CRP and homocysteine and with lower ICAM1, IL1R1, IL6, TNFα and NFκB1 gene expression in PBMC (P for trend ICAM1, TNFα and NFκB1 gene expression in PBMC showed a descending trend as increased fiber intake (>19.5 g/d from fruits and vegetables (P for trend 11.8 mmol/d of dietary total antioxidant capacity showed lower plasma CRP and mRNA values of ICAM1, IL1R1, IL6, TNFα and NFκB1 genes (P for trend Conclusion A higher fruit and vegetable consumption was independently associated not only with reduced CRP and homocysteine concentrations but also with a lower mRNA expression in PBMC of some relevant proinflammatory markers in healthy young adults.

  19. Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression.

    Science.gov (United States)

    Jayaraj, Jayaraman; Muthukrishnan, Subbaratnam; Liang, George H

    2004-07-01

    Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35-40 x 10(-6). The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.

  20. CplexA: a Mathematica package to study macromolecular-assembly control of gene expression

    OpenAIRE

    Vilar, J. M. G.; Saiz, L

    2010-01-01

    Summary: Macromolecular assembly vertebrates essential cellular processes, such as gene regulation and signal transduction. A major challenge for conventional computational methods to study these processes is tackling the exponential increase of the number of configurational states with the number of components. CplexA is a Mathematica package that uses functional programming to efficiently compute probabilities and average properties over such exponentially large number of states from the en...

  1. Technical note: identification of reference genes for gene expression studies in different bovine tissues focusing on different fat depots.

    Science.gov (United States)

    Saremi, B; Sauerwein, H; Dänicke, S; Mielenz, M

    2012-06-01

    Selection of stable reference genes (REF) is important in real-time PCR data normalization. Bovine tissues such as the mammary gland, liver, muscle, and s.c. fat from the tail head have been thoroughly explored for stable REF, whereas fewer reports exist for other fat depots. Therefore, a suitable combination of REF was tested for different tissues of dairy cattle. Holstein dairy heifers (n = 25) were supplemented (100 g/d) with a control fat (n = 15) without conjugated linoleic acids or with rumen-protected conjugated linoleic acids (n = 10) from the day of calving until slaughter at 1, 42, or 105 d postpartum (n = 5, 10, and 10, respectively). Samples from 6 fat depots (omental, mesenterial, retroperitoneal, s.c. tail head, s.c. withers, and s.c. sternum), liver, semitendinosus muscle, and mammary gland were collected. The REF mRNA were quantified and their stability was analyzed using geNorm(plus). The 3 most stable REF in individual fat tissues and muscle were EMD (emerin), POLR2A (RNA polymerase II), and LRP10 (lipoprotein receptor-related protein 10); in mammary gland were MARVELD1 (marvel domain containing 1), EMD, and LRP10; and in liver were HPCAL1 (hippocalcin-like 1), LRP10, and EIF3K (Eukaryotic translation initiation factor 3). The 3 most stable REF in s.c. fat were EMD, LRP10, and EIF3K; in visceral fat were POLR2A, LRP10, and MARVELD1; and for all 6 adipose tissues were LRP10, EIF3K, and MARVELD1. When the mammary gland was added to the 6 adipose depots, at least 5 REF (LRP10, POLR2A, EIF3K, MARVELD1, and HPCAL1) were needed to reach the threshold of 0.15. Addition of liver to the above-mentioned tissues increased the V value. The data improve the comparison of gene expression between different fat depots. In each case, GAPDH had the lowest stability value.

  2. First study on gene expression of cement proteins and potential adhesion-related genes of a membranous-based barnacle as revealed from Next-Generation Sequencing technology

    KAUST Repository

    Lin, Hsiu Chin

    2013-12-12

    This is the first study applying Next-Generation Sequencing (NGS) technology to survey the kinds, expression location, and pattern of adhesion-related genes in a membranous-based barnacle. A total of 77,528,326 and 59,244,468 raw sequence reads of total RNA were generated from the prosoma and the basis of Tetraclita japonica formosana, respectively. In addition, 55,441 and 67,774 genes were further assembled and analyzed. The combined sequence data from both body parts generates a total of 79,833 genes of which 47.7% were shared. Homologues of barnacle cement proteins - CP-19K, -52K, and -100K - were found and all were dominantly expressed at the basis where the cement gland complex is located. This is the main area where transcripts of cement proteins and other potential adhesion-related genes were detected. The absence of another common barnacle cement protein, CP-20K, in the adult transcriptome suggested a possible life-stage restricted gene function and/or a different mechanism in adhesion between membranous-based and calcareous-based barnacles. © 2013 © 2013 Taylor & Francis.

  3. Htr2a gene and 5-HT2A receptor expression in the cerebral cortex studied using genetically modified mice

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    Rodrigo Andrade

    2010-08-01

    Full Text Available Serotonin receptors of the 5-HT2A subtype are robustly expressed in the cerebral cortex where they have been implicated in the pathophysiology and therapeutics of mental disorders and the actions of hallucinogens. Much less is known, however, about the specific cell types expressing 5-HT2A receptors in cortex. In the current study we use immunohistochemical and electrophysiological approaches in genetically modified mice to address the expression of the Htr2a gene and 5-HT2A receptors in cortex. We first use an EGFP expressing BAC transgenic mice and identify three main Htr2A gene expressing neuronal populations in cortex. The largest of these cell populations corresponds to layer V pyramidal cells of the anterior cortex, followed by GABAergic interneurons of the middle layers, and nonpyramidal cells of the subplate/Layer VIb. We then use 5-HT2A receptor knockout mice to identify an antibody capable of localizing 5-HT2A receptors in brain and use it to map these receptors. We find strong laminar expression of 5-HT2A receptors in cortex, especially along a diffuse band overlaying layer Va. This band exhibits a strong anteroposterior gradient that closely matches the localization of Htr2A expressing pyramidal cells of layer V. Finally we use electrophysiological and immunohistochemical approaches to show that most, but not all, GABAergic interneurons of the middle layers are parvalbumin expressing Fast-spiking interneurons and that these cells are depolarized and excited by serotonin, most likely through the activation of 5-HT2A receptors. These results clarify and extend our understanding of the cellular distribution of 5-HT2A receptors in the cerebral cortex.

  4. Analysis of functional and pathway association of differential co-expressed genes: a case study in drug addiction.

    Science.gov (United States)

    Li, Zi-hui; Liu, Yu-feng; Li, Ke-ning; Duanmu, Hui-zi; Chang, Zhi-qiang; Li, Zhen-qi; Zhang, Shan-zhen; Xu, Yan

    2012-02-01

    Drug addiction has been considered as a kind of chronic relapsing brain disease influenced by both genetic and environmental factors. At present, many causative genes and pathways related to diverse kinds of drug addiction have been discovered, while less attention has been paid to common mechanisms shared by different drugs underlying addiction. By applying a co-expression meta-analysis method to mRNA expression profiles of alcohol, cocaine, heroin addicted and normal samples, we identified significant gene co-expression pairs. As co-expression networks of drug group and control group constructed, associated function term pairs and pathway pairs reflected by co-expression pattern changes were discovered by integrating functional and pathway information respectively. The results indicated that respiratory electron transport chain, synaptic transmission, mitochondrial electron transport, signal transduction, locomotory behavior, response to amphetamine, negative regulation of cell migration, glucose regulation of insulin secretion, signaling by NGF, diabetes pathways, integration of energy metabolism, dopamine receptors may play an important role in drug addiction. In addition, the results can provide theory support for studies of addiction mechanisms.

  5. Gene expression profile of sprinter's muscle.

    Science.gov (United States)

    Yoshioka, M; Tanaka, H; Shono, N; Shindo, M; St-Amand, J

    2007-12-01

    We have characterized the global gene expression profile in left vastus lateralis muscles of sprinters and sedentary men. The gene expression profile was analyzed by using serial analysis of gene expression (SAGE) method. The abundantly expressed transcripts in the sprinter's muscle were mainly involved in contraction and energy metabolism, whereas six transcripts were corresponding to potentially novel transcripts. Thirty-eight transcripts were differentially expressed between the sprinter and sedentary individuals. Moreover, sprinters showed higher expressions of both uncharacterized and potentially novel transcripts. Sprinters also highly expressed seven transcripts, such as glycine-rich protein, myosin heavy polypeptide (MYH) 2, expressed sequence tag similar to (EST) fructose-bisphosphate aldolase 1 isoform A (ALDOA), glyceraldehyde-3-phosphate dehydrogenase and ATP synthase F0 subunit 6. On the other hand, 20 transcripts such as MYH1, tropomyosin 2 and 3, troponin C slow, C2 fast, I slow, T1 slow and T3 fast, myoglobin, creatine kinase, ALDOA, glycogen phosphorylase, cytochrome c oxidase II and III, and NADH dehydrogenase 1 and 2 showed lower expression levels in the sprinters than the sedentary controls. The current study has characterized the global gene expressions in sprinters and identified a number of transcripts that can be subjected to further mechanistic analysis.

  6. Zipf's Law in Gene Expression

    CERN Document Server

    Furusawa, C; Furusawa, Chikara; Kaneko, Kunihiko

    2002-01-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1, i.e., they obey Zipf's law. Furthermore, by simulations of a simple model with an intra-cellular reaction network, we found that Zipf's law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  7. Zipf's Law in Gene Expression

    Science.gov (United States)

    Furusawa, Chikara; Kaneko, Kunihiko

    2003-02-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1; i.e., they obey Zipf’s law. Furthermore, by simulations of a simple model with an intracellular reaction network, we found that Zipf’s law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  8. Digital gene expression signatures for maize development.

    Science.gov (United States)

    Eveland, Andrea L; Satoh-Nagasawa, Namiko; Goldshmidt, Alexander; Meyer, Sandra; Beatty, Mary; Sakai, Hajime; Ware, Doreen; Jackson, David

    2010-11-01

    Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect the determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression profiles using Illumina's high-throughput sequencing technology and the newly assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in axillary meristem determinacy. The RA3 gene encodes a trehalose-6-phosphate phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of digital gene expression libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20- to 21-nucleotide reads with frequencies spanning 4 orders of magnitude. Unique sequence tags were anchored to 3'-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of nonredundant signature tags to the maize genome, which associated with 37,117 gene models and unannotated regions of expression. In total, 66% of genes were detected by at least nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) in functional analyses of differentially expressed maize genes. Results from this study provide a basis for the analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.

  9. Expression of polarity genes in human cancer.

    Science.gov (United States)

    Lin, Wan-Hsin; Asmann, Yan W; Anastasiadis, Panos Z

    2015-01-01

    Polarity protein complexes are crucial for epithelial apical-basal polarity and directed cell migration. Since alterations of these processes are common in cancer, polarity proteins have been proposed to function as tumor suppressors or oncogenic promoters. Here, we review the current understanding of polarity protein functions in epithelial homeostasis, as well as tumor formation and progression. As most previous studies focused on the function of single polarity proteins in simplified model systems, we used a genomics approach to systematically examine and identify the expression profiles of polarity genes in human cancer. The expression profiles of polarity genes were distinct in different human tissues and classified cancer types. Additionally, polarity expression profiles correlated with disease progression and aggressiveness, as well as with identified cancer types, where specific polarity genes were commonly altered. In the case of Scribble, gene expression analysis indicated its common amplification and upregulation in human cancer, suggesting a tumor promoting function.

  10. WT1 gene expression as a prognostic marker in advanced serous epithelial ovarian carcinoma: an immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Leetanaporn Roengsak

    2006-04-01

    Full Text Available Abstract Background WT1 is a tumor suppressor gene responsible for Wilms' tumor. WT1 reactivity is limited to ovarian serous carcinomas. Recent studies have shown that WT1 plays an important role in the progression of disease and indicates a poorer prognosis of human malignancies such as acute myeloid leukemia and breast cancer. The aims of this study were to determine the survival and recurrence-free survival of women with advanced serous epithelial ovarian carcinoma in relation to WT1 gene expression. Methods The study accrued women over an 18-year period, from 1987–2004. During the study period, 163 patients were diagnosed with advanced serous epithelial ovarian carcinoma and had undergone complete post-operative chemotherapy, but the final study group comprised 99 patients. The records of these women were reviewed and the paraffin-embedded tissue of these women stained with WT1 immunostaining. Survival analysis was performed using Kaplan-Meier and Cox regression methods. Results Fifty patients showed WT1 staining and forty-nine did not. Five-year survival of non-staining and staining groups were 39.4% and 10.7% (p Conclusion This study suggests that expression of WT1 gene may be indicative of an unfavorable prognosis in patients with advanced serous epithelial ovarian carcinoma.

  11. In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.

    Science.gov (United States)

    Dudczig, Stefanie; Currie, Peter D; Poggi, Lucia; Jusuf, Patricia R

    2017-03-22

    The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

  12. Foxp3 gene expression in oral lichen planus: a clinicopathological study.

    Science.gov (United States)

    Lei, Lei; Zhan, Lihua; Tan, Weixia; Chen, Shaohua; Li, Yangqiu; Reynolds, Mark

    2014-03-01

    CD4+CD25+ Forkhead‑box protein 3 (Foxp3+) regulatory T cells are important in oral lichen planus (OLP). The present study aimed to investigate Foxp3 expression in CD4+CD25+ T cells of peripheral blood mononuclear cells and oral lesions in patients diagnosed with OLP, who were grouped as OLP subtype, duration and relapse. Using quantitative polymerase chain reaction (qPCR), western blotting and immunohistochemistry, Foxp3 expression levels in explants of oral lesions and CD4+CD25+ T cells from 32 patients with OLP were measured and compared, with 10 healthy subjects as the control group. Foxp3 mRNA expression levels in the explants of oral lesions and circulating CD4+CD25+ T cells in patients with OLP were significantly higher than those in the control group (Pexpression was significantly lower in circulating CD4+CD25+ T cells and tissue explants compared to patients with reticular lesions (P1 year or with a history of relapse (Pexpression in OLP tissues was 36.24±18.92 and 10.44±6.51% in normal oral mucosa (P=0.019). Atrophic/erosive OLP lesions showed a higher proportion of Foxp3‑expressing cells than that of reticular OLP lesions (Pexpression in patients with OLP is associated with the severity and duration of the disorder, suggesting altered immune suppression in the development, clinical course and responsiveness to treatment.

  13. Study on Tibetan Chicken embryonic adaptability to chronic hypoxia by revealing differential gene expression in heart tissue

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Oxygen concentration is essential for appropriate metabolism.Hypoxia can exert a significant impact on physiological alteration of the cell and organism.Tibetan Chicken(Gallus gallus) is a Chinese indigenous breed inhabiting in Tibetan areas,which is also a chicken breed living at high altitude for the longest time in the world.It has developed an adaptive mechanism to hypoxia,which is demonstrated by that Tibetan Chicken has much higher hatchability than low-land chicken breeds in high-altitude areas of Tibet.In the present study,Tibetan Chicken fertilized full sib eggs were incubated up to Hamburger-Hamilton stage 43 under 13% and 21% oxygen concentration,respectively.Shouguang Chicken and Dwarf Recessive White Chicken were used as control groups.The hearts in all of the 3 chicken breeds under hypoxic and normoxic conditions were isolated and hybridized to Genechip Chicken Genome Array to study molecular mechanisms underlying the adaptation to high altitude of Tibetan Chicken.As a result,50 transcripts highly expressed in hypoxia are screened out.Among up-regulated genes,some are involved in the gene ontology(GO) such as cell growth,cell difference,muscle contraction and signal transduction.However,the expression levels of 21 transcripts are lower in hypoxia than those in normoxia.Some down-regulated genes take part in cell communication,ion transport,protein amino acid phosphorylation and signal transduction.Interestingly,gene enrichment analyses of these differential gene expressions are mainly associated with immune system response and ion channel activity in response to stimulus.Moreover,the transcriptional expression profiles analyzed by hierarchical clustering and CPP-SOM software in all of the 3 different chicken breeds revealed that Tibetan Chicken is much closely related to Shouguang Chicken rather than Dwarf Recessive White Chicken.In addition,12 transcripts of Tibetan Chicken breed-specific expressed genes were identified,which seem to result in a

  14. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis.

    Science.gov (United States)

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis.

  15. Effect of irradiation on gene expression of rat liver adhesion molecules. In vivo and in vitro studies

    Energy Technology Data Exchange (ETDEWEB)

    Moriconi, Federico; Malik, Ihtzaz; Ahmad, Ghayyor; Dudas, Joszef; Ramadori, Giuliano [Dept. of Gastroenterology and Endocrinology, Goettingen Univ. (Germany); Rave-Fraenk, Margret; Vorwerk, Hilke; Hille, Andrea; Hess, Clemens Friedrich; Christiansen, Hans [Dept. of Radiotherapy, Goettingen Univ. (Germany)

    2009-07-15

    Background and purpose: Migration of leukocytes into tissue is a key element of innate and adaptive immunity. An animal study showed that liver irradiation, in spite of induction of chemokine gene expression, does not lead to recruitment of leukocytes into the parenchyma. The aim of this study was to analyze gene expression of adhesion molecules, which mediate leukocyte recruitment into organs, in irradiated rat liver in vivo and rat hepatocytes in vitro. Material and methods: Rat livers in vivo were irradiated selectively at 25 Gy. Isolated hepatocytes in vitro were irradiated at 8 Gy. RNA extracted within 48 h after irradiation in vivo and in vitro was analyzed by real-time PCR (polymerase chain reaction) and Northern blot. Adhesion molecule concentration in serum was measured by ELISA (enzyme-linked immunosorbent assay). Cryostat sections of livers were used for immunohistology. Results: Significant radiation-induced increase of ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), JAM-1 (junctional adhesion molecule-1), {beta}{sub 1}-integrin, {beta}{sub 2}-integrin, E-cadherin, and P-selectin gene expression could be detected in vivo, while PECAM-1 (platelet-endothelial cell adhesion molecule-1) gene expression remained unchanged. In vitro, {beta}{sub 1}-integrin, JAM-1, and ICAM-2 showed a radiation-induced increased expression, whereas the levels of P-selectin, ICAM-1, PECAM-1, VCAM-1, Madcam-1 (mucosal addressin cell adhesion molecule-1), {beta}{sub 2}-integrin, and E-cadherin were downregulated. However, incubation of irradiated hepatocytes with either tumor necrosis factor-(TNF-){alpha}, interleukin-(IL-)1{beta}, or IL-6 plus TNF-{alpha} led to an upregulation of P-selectin, ICAM-1 and VCAM-1. Conclusion: The findings suggest that liver irradiation modulates gene expression of the main adhesion molecules in vivo and in cytokine-activated hepatocytes, with the exception of PECAM-1. This may be one reason for the lack of

  16. Dissecting inflammatory complications in critically injured patients by within-patient gene expression changes: a longitudinal clinical genomics study.

    Directory of Open Access Journals (Sweden)

    Keyur H Desai

    2011-09-01

    Full Text Available BACKGROUND: Trauma is the number one killer of individuals 1-44 y of age in the United States. The prognosis and treatment of inflammatory complications in critically injured patients continue to be challenging, with a history of failed clinical trials and poorly understood biology. New approaches are therefore needed to improve our ability to diagnose and treat this clinical condition. METHODS AND FINDINGS: We conducted a large-scale study on 168 blunt-force trauma patients over 28 d, measuring ∼400 clinical variables and longitudinally profiling leukocyte gene expression with ∼800 microarrays. Marshall MOF (multiple organ failure clinical score trajectories were first utilized to organize the patients into five categories of increasingly poor outcomes. We then developed an analysis framework modeling early within-patient expression changes to produce a robust characterization of the genomic response to trauma. A quarter of the genome shows early expression changes associated with longer-term post-injury complications, captured by at least five dynamic co-expression modules of functionally related genes. In particular, early down-regulation of MHC-class II genes and up-regulation of p38 MAPK signaling pathway were found to strongly associate with longer-term post-injury complications, providing discrimination among patient outcomes from expression changes during the 40-80 h window post-injury. CONCLUSIONS: The genomic characterization provided here substantially expands the scope by which the molecular response to trauma may be characterized and understood. These results may be instrumental in furthering our understanding of the disease process and identifying potential targets for therapeutic intervention. Additionally, the quantitative approach we have introduced is potentially applicable to future genomics studies of rapidly progressing clinical conditions. TRIAL REGISTRATION: ClinicalTrials.gov NCT00257231

  17. Widespread ectopic expression of olfactory receptor genes

    Directory of Open Access Journals (Sweden)

    Yanai Itai

    2006-05-01

    Full Text Available Abstract Background Olfactory receptors (ORs are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information.

  18. A database study that identifies genes whose expression correlates, negatively or positively, with 5-year survival of cancer patients

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2007-01-01

    A published microarray gene expression database containing data on 174 tumor samples from ten tissues was mined, enabling the identification of classes of genes whose expression correlates significantly with the intractability, or tractability, to therapy of tumors derived from such tissues...

  19. Prospective study on the expression of cancer testis genes and antibody responses in 100 consecutive patients with primary breast cancer.

    NARCIS (Netherlands)

    Mischo, A.; Kubuschok, B.; Ertan, K.; Preuss, K.D.; Romeike, B.; Regitz, E.; Schormann, C.; Bruijn, D.R.H. de; Wadle, A.; Neumann, F.; Schmidt, W.; Renner, C.; Pfreundschuh, M.

    2006-01-01

    To determine the expression of cancer testis (CT) genes and antibody responses in a nonselected population of patients with primary breast cancer, we investigated the composite expression of 11 CT genes by RT-PCR in fresh biopsies of 100 consecutive cases of primary breast carcinoma and by immunohis

  20. A practical platform for blood biomarker study by using global gene expression profiling of peripheral whole blood.

    Directory of Open Access Journals (Sweden)

    Ze Tian

    Full Text Available BACKGROUND: Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study. METHODS AND FINDINGS: We applied peripheral whole blood samples with globin reduction and performed genome-wide transcriptome analysis using Illumina BeadChips. Real-time PCR was subsequently used to evaluate the quality of array data and elucidate the mode in which hemoglobin interferes in gene expression profiling. We demonstrated that, when applied in the context of standard microarray processing procedures, globin reduction results in a consistent and significant increase in the quality of beadarray data. When compared to their pre-globin reduction counterparts, post-globin reduction samples show improved detection statistics, lowered variance and increased sensitivity. More importantly, gender gene separation is remarkably clearer in post-globin reduction samples than in pre-globin reduction samples. Our study suggests that the poor data obtained from pre-globin reduction samples is the result of the high concentration of hemoglobin derived from red blood cells either interfering with target mRNA binding or giving the pseudo binding background signal. CONCLUSION: We therefore recommend the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips as the practical approach for biomarker study.

  1. Perspectives: Gene Expression in Fisheries Management

    Science.gov (United States)

    Nielsen, Jennifer L.; Pavey, Scott A.

    2010-01-01

    Functional genes and gene expression have been connected to physiological traits linked to effective production and broodstock selection in aquaculture, selective implications of commercial fish harvest, and adaptive changes reflected in non-commercial fish populations subject to human disturbance and climate change. Gene mapping using single nucleotide polymorphisms (SNPs) to identify functional genes, gene expression (analogue microarrays and real-time PCR), and digital sequencing technologies looking at RNA transcripts present new concepts and opportunities in support of effective and sustainable fisheries. Genomic tools have been rapidly growing in aquaculture research addressing aspects of fish health, toxicology, and early development. Genomic technologies linking effects in functional genes involved in growth, maturation and life history development have been tied to selection resulting from harvest practices. Incorporating new and ever-increasing knowledge of fish genomes is opening a different perspective on local adaptation that will prove invaluable in wild fish conservation and management. Conservation of fish stocks is rapidly incorporating research on critical adaptive responses directed at the effects of human disturbance and climate change through gene expression studies. Genomic studies of fish populations can be generally grouped into three broad categories: 1) evolutionary genomics and biodiversity; 2) adaptive physiological responses to a changing environment; and 3) adaptive behavioral genomics and life history diversity. We review current genomic research in fisheries focusing on those that use microarrays to explore differences in gene expression among phenotypes and within or across populations, information that is critically important to the conservation of fish and their relationship to humans.

  2. Sex differentiation in Atlantic cod (Gadus morhua L.: morphological and gene expression studies

    Directory of Open Access Journals (Sweden)

    Haugen Trine

    2012-06-01

    Full Text Available Abstract Background In differentiated gonochoristic species, a bipotential gonad develops into an ovary or testis during sex differentiation. Knowledge about this process is necessary to improve methods for masculinizing genetically female Atlantic cod for the subsequent purpose of producing all-female populations. Methods Gonads were examined histologically in juveniles from 14 to 39 mm total body length (TL. Number and size of germ cells were determined in a subset of the samples. Relevant genes were cloned, and mRNA levels determined by qPCR of amh, cyp19a1a; dax1 (nr0b2; shp (nr0b2a and sox9b in a mixed-sex and an all-female population ranging from 12–49 mm TL. Results Individuals between 14–20 mm TL could be separated in two subgroups based on gonad size and germ cell number. Ovarian cavity formation was observed in some individuals from 18–20 mm TL. The mixed sex population displayed bimodal expression patterns as regards cyp19a1a (starting at 12 mm TL and amh (starting at 20 mm TL mRNA levels. After approximately 30 mm TL, cyp19a1a and amh displayed a gradual increase in both sexes. No apparent, sex-dependent expression patterns were found for dax1, shp or sox9b transcripts. However, shp levels were high until the larvae reached around 35 mm TL and then dropped to low levels, while dax1 remained low until 35 mm TL, and then increased sharply. Conclusions The morphological sex differentiation in females commenced between 14–20 mm TL, and ovarian cavities were evident by 18–20 mm TL. Testis development occurred later, and was morphologically evident after 30 mm TL. This pattern was corroborated with sexually dimorphic expression patterns of cyp19a1a from 12–13 mm TL, and a male-specific increase in amh from 20 mm TL.

  3. Adaptation of yeasts Saccharomyces cerevisiae and Brettanomyces bruxellensis to winemaking conditions: a comparative study of stress genes expression.

    Science.gov (United States)

    Nardi, Tiziana; Remize, Fabienne; Alexandre, Hervé

    2010-10-01

    Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. Like Saccharomyces cerevisiae, it is well adapted to winemaking, but molecular pathways involved in this acclimatization are still unknown. In this work, we report a time-scale comparison between the two yeasts coping with alcoholic fermentation. Orthologs of some well-characterized stress genes of S. cerevisiae were searched by sequence alignment in the Dekkera/Brettanomyces partial genome; nine genes were finally selected on the basis on their similarity and involvement in adaptation to wine. Transcript analysis during a model grape juice fermentation indicates that a subset of genes (i.e., ATP1, ERG6, VPS34) shows peculiar expression patterns in Brettanomyces bruxellensis but also that some common regulations of stress response exist between the two yeasts, although with different timing (i.e., for MSN4, SNF1, HSP82, NTH1). This suggests that B. bruxellensis efficient survival in such challenging conditions is due to mechanisms unique to it, together with a conserved yeast stress response. This study, although limited by the poor genetic data available on B. bruxellensis, provides first insights into its gene expression remodeling in winemaking and opens new frames for further investigations.

  4. Validation of housekeeping genes for the normalization of RT-qPCR expression studies in oral squamous cell carcinoma cell line treated by 5 kinds of chemotherapy drugs.

    Science.gov (United States)

    Song, W; Zhang, W H; Zhang, H; Li, Y; Zhang, Y; Yin, W; Yang, Q

    2016-11-30

    Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT-qPCR data. The key of this method is to identify an applicable internal control gene because the usage of different internal control genes may lead to distinct conclusions. Herein, we report the validation of 12 common housekeeping genes for RT-qPCR for gene expression analysis in the Oral squamous cell carcinoma (OSCC) cell line (KB and Tca-8113) treated by 5 kinds of Chemotherapy Drugs. The gene expression stability and applicability of the 12 housekeeping gene candidates were determined using the geNorm, NormFinder, and BestKeeper software programs. Comprehensive analyzing the results of the three software, ALAS1/GAPDH, ALAS1 and GUSB were suggested to be the most stable candidate genes for the study of both KB and Tca-8113 cell line together, KB cell line, and Tca-8113 cell line, respectively. This study provides useful information to normalize gene expression accurately for the investigation of target gene profiling in cell lines of OSCC. Further clarification of tumor molecular expression markers with our recommended housekeeping genes may improve the accuracy of diagnosis and estimation of prognostic factors as well as provide novel personalized treatments for OSCC patients.

  5. Lymphocyte differentiation in sea bass thymus: CD4 and CD8-alpha gene expression studies.

    Science.gov (United States)

    Picchietti, Simona; Guerra, Laura; Buonocore, Francesco; Randelli, Elisa; Fausto, Anna Maria; Abelli, Luigi

    2009-07-01

    Different developmental stages (from eggs to 1-year-old juveniles) of the teleost fish Dicentrarchus labrax (L.) were assayed for CD4 gene expression. RT-PCR revealed the appearance of CD4 transcripts in post-larvae from 51 days post-hatching (dph). This finding overlaps the first detection of CD8-alpha mRNA. Real-time PCR with specific primers quantified CD4, CD8-alpha and TCR-beta transcripts in larvae and post-larvae (25, 51, 75 and 92 dph) and 1-year-old thymus. At 92 dph, TcR-beta and CD8-alpha transcripts were significantly higher (P overlap, except in the medulla, where CD4(+) thymocytes were isolated, while CD8-alpha(+) ones mainly arranged in cords. These results provide new information about the thymic compartmentalization and lymphocyte differentiation pathways in a teleost, almost demonstrating that double negative thymocytes fill the cortex giving rise to further selection in the medulla.

  6. Regulation of immunoglobulin gene rearrangement and expression.

    Science.gov (United States)

    Taussig, M J; Sims, M J; Krawinkel, U

    1989-05-01

    The molecular genetic events leading to Ig expression and their control formed the topic of a recent EMBO workshop. This report by Michael Taussig, Martin Sims and Ulrich Krawinkel discusses contributions dealing with genes expressed in early pre-B cells, the mechanism of rearrangement, aberrant rearrangements seen in B cells of SCID mice, the feedback control of rearrangement as studied in transgenic mice, the control of Ig expression at the transcriptional and post-transcriptional levels, and class switching.

  7. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa. Not...

  8. Gene expression of the endolymphatic sac.

    Science.gov (United States)

    Friis, Morten; Martin-Bertelsen, Tomas; Friis-Hansen, Lennart; Winther, Ole; Henao, Ricardo; Sørensen, Mads Sølvsten; Qvortrup, Klaus

    2011-12-01

    The endolymphatic sac is part of the membranous inner ear and is thought to play a role in the fluid homeostasis and immune defense of the inner ear; however, the exact function of the endolymphatic sac is not fully known. Many of the detected mRNAs in this study suggest that the endolymphatic sac has multiple and diverse functions in the inner ear. The objective of this study was to provide a comprehensive review of the genes expressed in the endolymphatic sac in the rat and perform a functional characterization based on measured mRNA abundance. Microarray technology was used to investigate the gene expression of the endolymphatic sac with the surrounding dura. Characteristic and novel endolymphatic sac genes were determined by comparing with expressions in pure dura. In all, 463 genes were identified specific for the endolymphatic sac. Functional annotation clustering revealed 29 functional clusters.

  9. Evaluation of the use of the polyubiquitin genes, Ubi4 and Ubi10 as reference genes for expression studies in Brachypodium distachyon.

    Directory of Open Access Journals (Sweden)

    John P Chambers

    Full Text Available BACKGROUND: Brachypodium distachyon is emerging as the model plant for temperate grass research and the genome of the community line Bd21 has been sequenced. Additionally, techniques have been developed for Agrobacterium-mediated transformation for the generation of T-DNA insertional lines. Recently, it was reported that expression of the polyubiquitin genes, Ubi4 and Ubi10 are stable in different tissues and growth hormone-treated plant samples, leading to the conclusion that both Ubi4 and Ubi10 are good reference genes for normalization of gene expression data using real-time, quantitative PCR (qPCR. PRINCIPAL FINDINGS: Mining of the Joint Genome Institute (JGI 8X Brachypodium distachyon genome assembly showed that Ubi4 and Ubi10 share a high level of sequence identity (89%, and in silico analyses of the sequences of Ubi4 (Bradi3g04730 and Ubi10 (Bradi1g32860 showed that the primers used previously exhibit multiple binding sites within the coding sequences arising from the presence of tandem repeats of the coding regions. This can potentially result in over-estimation of steady-state levels of Ubi4 and Ubi10. Additionally, due to the high level of sequence identity between both genes, primers used previously for amplification of Ubi4 can bind to Ubi10 and vice versa, resulting in the formation of non-specific amplification products. CONCLUSIONS: The results from this study indicate that the primers used previously were not sufficiently robust and specific. Additionally, their use would result in over-estimation of the steady-state expression levels of Ubi4. Our results question the validity of using the previously proposed primer sets for qPCR amplification of Ubi4 and Ubi10. We demonstrate that primers designed to target the 3'-UTRs of Ubi4 and Ubi10 are better suited for real-time normalization of steady-state expression levels in Brachypodium distachyon.

  10. An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1

    Directory of Open Access Journals (Sweden)

    Alison S. Devonshire

    2016-06-01

    Full Text Available Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM-P103.1 was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis Working Group of the CCQM. It was coordinated by LGC (United Kingdom with the support of National Institute of Standards and Technology (USA and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals (‘measurement uncertainties’ were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and

  11. Study on Tibetan Chicken embryonic adaptability to chronic hypoxia by revealing differential gene expression in heart tissue

    Institute of Scientific and Technical Information of China (English)

    LI Mei; ZHAO ChunJiang

    2009-01-01

    Oxygen concentration is essential for appropriate metabolism. Hypoxia can exert a significant impact on physiological alteration of the cell and organism. Tibetan Chicken (Gallus gallus) is a Chinese in-digenous breed inhabiting in Tibetan areas, which is also a chicken breed living at high altitude for the longest time in the world. It has developed an adaptive mechanism to hypoxia, which is demonstrated by that Tibetan Chicken has much higher hatchability than low-land chicken breeds in high-altitude areas of Tibet. In the present study, Tibetan Chicken fertilized full sib eggs were incubated up to Ham-burger-Hamilton stage 43 under 13% and 21% oxygen concentration, respectively. Shouguang Chicken and Dwarf Recessive White Chicken were used as control groups. The hearts in all of the 3 chicken breeds under hypoxic and normoxic conditions were isolated and hybridized to GeneChip Chicken Genome Array to study molecular mechanisms underlying the adaptation to high altitude of Tibetan Chicken. As a result, 50 transcripts highly expressed in hypoxia are screened out. Among up-regulated genes, some are involved in the gone ontology (GO) such as cell growth, cell difference, muscle con-traction and signal transduction. However, the expression levels of 21 transcripts are lower in hypoxia than those in normoxia. Some down-regulated genes take part in cell communication, ion transport, protein amino acid phosphorylation and signal transduction. Interestingly, gene enrichment analyses of these differential gone expressions are mainly associated with immune system response and ion channel activity in response to stimulus. Moreover, the transcriptional expression profiles analyzed by hierarchical clustering and CPP-SOM software in all of the 3 different chicken breeds revealed that TI-betan Chicken is much closely related to Shouguang Chicken rather than Dwarf Recessive White Chicken. In addition, 12 transcripts of Tibetan Chicken breed-specific expressed genes were

  12. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.

    2013-07-18

    The modeling of gene networks from transcriptional expression data is an important tool in biomedical research to reveal signaling pathways and to identify treatment targets. Current gene network modeling is primarily based on the use of Gaussian graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which generate counts of mRNAtranscripts in cell samples.We propose a generalized linear model to fit the discrete gene expression data and assume that the log ratios of the mean expression levels follow a Gaussian distribution.We restrict the gene network structures to decomposable graphs and derive the graphs by selecting the covariance matrix of the Gaussian distribution with the hyper-inverse Wishart priors. Furthermore, we incorporate prior network models based on gene ontology information, which avails existing biological information on the genes of interest. We conduct simulation studies to examine the performance of our discrete graphical model and apply the method to two real datasets for gene network inference. © The Author 2013. Published by Oxford University Press. All rights reserved.

  13. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  14. Integrated analyses of gene expression and genetic association studies in a founder population

    Science.gov (United States)

    Cusanovich, Darren A.; Caliskan, Minal; Billstrand, Christine; Michelini, Katelyn; Chavarria, Claudia; De Leon, Sherryl; Mitrano, Amy; Lewellyn, Noah; Elias, Jack A.; Chupp, Geoffrey L.; Lang, Roberto M.; Shah, Sanjiv J.; Decara, Jeanne M.; Gilad, Yoav; Ober, Carole

    2016-01-01

    Genome-wide association studies (GWASs) have become a standard tool for dissecting genetic contributions to disease risk. However, these studies typically require extraordinarily large sample sizes to be adequately powered. Strategies that incorporate functional information alongside genetic associations have proved successful in increasing GWAS power. Following this paradigm, we present the results of 20 different genetic association studies for quantitative traits related to complex diseases, conducted in the Hutterites of South Dakota. To boost the power of these association studies, we collected RNA-sequencing data from lymphoblastoid cell lines for 431 Hutterite individuals. We then used Sherlock, a tool that integrates GWAS and expression quantitative trait locus (eQTL) data, to identify weak GWAS signals that are also supported by eQTL data. Using this approach, we found novel associations with quantitative phenotypes related to cardiovascular disease, including carotid intima-media thickness, left atrial volume index, monocyte count and serum YKL-40 levels. PMID:26931462

  15. Neighboring Genes Show Correlated Evolution in Gene Expression

    Science.gov (United States)

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  16. Gene expression profiling: can we identify the right target genes?

    Directory of Open Access Journals (Sweden)

    J. E. Loyd

    2008-12-01

    Full Text Available Gene expression profiling allows the simultaneous monitoring of the transcriptional behaviour of thousands of genes, which may potentially be involved in disease development. Several studies have been performed in idiopathic pulmonary fibrosis (IPF, which aim to define genetic links to the disease in an attempt to improve the current understanding of the underlying pathogenesis of the disease and target pathways for intervention. Expression profiling has shown a clear difference in gene expression between IPF and normal lung tissue, and has identified a wide range of candidate genes, including those known to encode for proteins involved in extracellular matrix formation and degradation, growth factors and chemokines. Recently, familial pulmonary fibrosis cohorts have been examined in an attempt to detect specific genetic mutations associated with IPF. To date, these studies have identified families in which IPF is associated with mutations in the gene encoding surfactant protein C, or with mutations in genes encoding components of telomerase. Although rare and clearly not responsible for the disease in all individuals, the nature of these mutations highlight the importance of the alveolar epithelium in disease pathogenesis and demonstrate the potential for gene expression profiling in helping to advance the current understanding of idiopathic pulmonary fibrosis.

  17. Study on the Effects of Losartan on Cardiomyocyte Apoptosis and Gene Expression After Ischemia and Reperfusion in vivo in Rats

    Institute of Scientific and Technical Information of China (English)

    ZHANG Dongqing; YANG Liming; LIU Zhengxiang; MI Shizan

    2000-01-01

    In order to study the effects of losartan on cardiomyocyte apoptosis following ischemia (0.5 h) and reperfusion (48 h) in vivo and bcl-2 and bax gene expression, TUNEL staining method, immunohistochemistry and in situ hybridization histochemistry (ISHH) were used to monitor the apoptotic cells, mRNA and protein of gene expression, respectively. Image processing system was used to quantitively dispose the positive metric substance of both immunohistochemistry and ISHH through the average optical density (OD) value. The number of the apoptotic cells were 38±9 (control group), 0-1 (sham operation group) and 9±4 (losartan-treated group) in each visual field respectively with the difference among the groups being significant (P<0.001). OD values of bcl-2 (ISHH) were 0.07425±0.02029 (control group), 0.05961±0.009932 (sham operation group) and 0. 07619±0.01445 (losartan-treated group) respectively,while OD values of bcl-2 (immunohistochemistry) were 0.1374 ±0.01367 (control group),0.08510±0.01862 (sham operation group) and 0.1252±0.02064 (losartan-treated group), bcl-2gene expression was increased significantly in the control group and losartan-treated group as compared with sham operation group (P<0.05). OD value of bax (immunohistochemistry) was09727± 0.02230 (control group), 0.06182±0.01430 (sham operation group) and 0.06213 ±0.01420 (losartan-treated group), bax gene expression was decreased very significantly in losartan-treated group and sham operation group as compared with control group (P<0. 001). Bcl-2/bax ratio was 1.413 (control group), 1.376 (sham operation group) and 2.016 (losartan-treated group) respectively. The results indicated that losartan might inhibit cardiomyocyte apoptosis following ischemia and reperfusion. The mechanism might be that bax gene expression was inhibited to increase bcl-2/bax ratio.

  18. Molecular characterization of the 14-3-3 gene family in rice and its expression studies under abiotic stress.

    Science.gov (United States)

    Yashvardhini, Niti; Bhattacharya, Saurav; Chaudhuri, Shubho; Sengupta, Dibyendu Narayan

    2017-09-27

    14-3-3 isoforms were relatively less conserved at the C-terminal region across plant groups. Both Os 14-3-3f and Os 14-3-3g were inducible with differential gene expression levels under different abiotic stress and developmental stages in sensitive and tolerant indica rice cultivars as confirmed both at transcript and protein level. Plant 14-3-3s has been well characterized to function in several signaling pathways, biotic as well as abiotic stress and nutrient metabolism. We attempted comprehensive analysis of 14-3-3 genes in different plant lineages such as green algae (Chlamydomonas reinhardtii), moss (Physcomitrella patens) and lycophyte (Selaginella moellendorffii), dicot Arabidopsis thaliana and monocot Oryza sativa sub sp. japonica at the gene and protein level. Sequence alignment results revealed that 14-3-3 isoforms were evolutionarily conserved across all taxa with variable C-terminal end. Phylogenetic analysis indicated that the majority of 14-3-3 isoforms in rice belong to the non-epsilon group that clustered separately from the dicot group. Segmental duplication event played a significant role in the expansion of both, Arabidopsis and rice, 14-3-3 isoforms as revealed by synteny studies. In silico gene expression using Massive Parallel Signature Sequencing and microarray analysis revealed that 14-3-3 isoforms have variable expression in different tissue types and under different abiotic stress regime in Arabidopsis and japonica rice. Both, semi-quantitative and qPCR results, confirmed that Os14-3-3f and Os14-3-3g were inducible under abiotic stress in lamina and roots of indica rice and relatively higher under salinity and cold stress in Nonabokra, under dehydration stress in N-22 and under exogenous ABA in IR-29 usually after 3-6 h of treatment. Both, 14-3-3f and 14-3-3g, were highly expressed in flag leaves, stems and panicles and mature roots. These results were further confirmed by immunoblot analysis of rice cultivars using Os14-3-3f antibody

  19. Comprehensive study of gene and microRNA expression related to epithelial-mesenchymal transition in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Betina Katz

    Full Text Available Prostate cancer is the most common cancer in men, and most patients have localized disease at the time of diagnosis. However, 4% already present with metastatic disease. Epithelial-mesenchymal transition is a fundamental process in carcinogenesis that has been shown to be involved in prostate cancer progression. The main event in epithelial-mesenchymal transition is the repression of E-cadherin by transcription factors, but the process is also regulated by microRNAs. The aim of this study was to analyze gene and microRNA expression involved in epithelial-mesenchymal transition in localized prostate cancer and metastatic prostate cancer cell lines and correlate with clinicopathological findings. We studied 51 fresh frozen tissue samples from patients with localized prostate cancer (PCa treated by radical prostatectomy and three metastatic prostate cancer cell lines (LNCaP, DU145, PC3. The expression of 10 genes and 18 miRNAs were assessed by real-time PCR. The patients were divided into groups according to Gleason score, pathological stage, preoperative PSA, biochemical recurrence, and risk group for correlation with clinicopathological findings. The majority of localized PCa cases showed an epithelial phenotype, with overexpression of E-cadherin and underexpression of the mesenchymal markers. MiRNA-200 family members and miRNAs 203, 205, 183, 373, and 21 were overexpressed, while miRNAs 9, 495, 29b, and 1 were underexpressed. Low-expression levels of miRNAs 200b, 30a, and 1 were significantly associated with pathological stage. Lower expression of miR-200b was also associated with a Gleason score ≥ 8 and shorter biochemical recurrence-free survival. Furthermore, low-expression levels of miR-30a and high-expression levels of Vimentin and Twist1 were observed in the high-risk group. Compared with the primary tumor, the metastatic cell lines showed significantly higher expression levels of miR-183 and Twist1. In summary, miRNAs 200b, 30a, 1, and

  20. Insulin gene: organisation, expression and regulation.

    Science.gov (United States)

    Dumonteil, E; Philippe, J

    1996-06-01

    Insulin, a major hormone of the endocrine pancreas, plays a key role in the control of glucose homeostasis. This review discusses the mechanisms of cell-specific expression and regulation of the insulin gene. Whereas expression is restricted to islet beta-cells in adults, the insulin gene is more widely expressed at several embryonic stages, although the role of extrapancreatic expression is still unclear. beta-cell-specific expression relies on the interactions of 5'-flanking sequence motifs of the promoter with a number of ubiquitous and islet-specific transcription factors. IEF1 and IPF-1, by their binding to the E and A boxes, respectively, of the insulin gene promoter, appear to be the major determinants of beta-cell-specific expression. IEF1 is a heterodimer of the basic helix-loop-helix family of transcription factors, whereas IPF-1 belongs to the homeodomain-containing family. beta-cell specific determinants are conserved throughout evolution, although the human insulin gene 5'-flanking sequence also contains a polymorphic minisatellite which is unique to primates and may play a role in insulin gene regulation. Glucose modulates insulin gene transcription, with multiple elements of the promoter involved in glucose responsiveness. Remarkably, IPF-1 and IEF1 are involved in both beta-cell-specific expression and glucose regulation of the insulin gene. cAMP also regulates insulin gene transcription through a CRE, in response to various hormonal stimuli. On the whole, recent studies have provided a better understanding of beta-cell differentiation and function.

  1. Application of multidisciplinary analysis to gene expression.

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xuefel (University of New Mexico, Albuquerque, NM); Kang, Huining (University of New Mexico, Albuquerque, NM); Fields, Chris (New Mexico State University, Las Cruces, NM); Cowie, Jim R. (New Mexico State University, Las Cruces, NM); Davidson, George S.; Haaland, David Michael; Sibirtsev, Valeriy (New Mexico State University, Las Cruces, NM); Mosquera-Caro, Monica P. (University of New Mexico, Albuquerque, NM); Xu, Yuexian (University of New Mexico, Albuquerque, NM); Martin, Shawn Bryan; Helman, Paul (University of New Mexico, Albuquerque, NM); Andries, Erik (University of New Mexico, Albuquerque, NM); Ar, Kerem (University of New Mexico, Albuquerque, NM); Potter, Jeffrey (University of New Mexico, Albuquerque, NM); Willman, Cheryl L. (University of New Mexico, Albuquerque, NM); Murphy, Maurice H. (University of New Mexico, Albuquerque, NM)

    2004-01-01

    Molecular analysis of cancer, at the genomic level, could lead to individualized patient diagnostics and treatments. The developments to follow will signal a significant paradigm shift in the clinical management of human cancer. Despite our initial hopes, however, it seems that simple analysis of microarray data cannot elucidate clinically significant gene functions and mechanisms. Extracting biological information from microarray data requires a complicated path involving multidisciplinary teams of biomedical researchers, computer scientists, mathematicians, statisticians, and computational linguists. The integration of the diverse outputs of each team is the limiting factor in the progress to discover candidate genes and pathways associated with the molecular biology of cancer. Specifically, one must deal with sets of significant genes identified by each method and extract whatever useful information may be found by comparing these different gene lists. Here we present our experience with such comparisons, and share methods developed in the analysis of an infant leukemia cohort studied on Affymetrix HG-U95A arrays. In particular, spatial gene clustering, hyper-dimensional projections, and computational linguistics were used to compare different gene lists. In spatial gene clustering, different gene lists are grouped together and visualized on a three-dimensional expression map, where genes with similar expressions are co-located. In another approach, projections from gene expression space onto a sphere clarify how groups of genes can jointly have more predictive power than groups of individually selected genes. Finally, online literature is automatically rearranged to present information about genes common to multiple groups, or to contrast the differences between the lists. The combination of these methods has improved our understanding of infant leukemia. While the complicated reality of the biology dashed our initial, optimistic hopes for simple answers from

  2. Concordance analysis of microarray studies identifies representative gene expression changes in Parkinson's disease: a comparison of 33 human and animal studies.

    Science.gov (United States)

    Oerton, Erin; Bender, Andreas

    2017-03-23

    As the popularity of transcriptomic analysis has grown, the reported lack of concordance between different studies of the same condition has become a growing concern, raising questions as to the representativeness of different study types, such as non-human disease models or studies of surrogate tissues, to gene expression in the human condition. In a comparison of 33 microarray studies of Parkinson's disease, correlation and clustering analyses were used to determine the factors influencing concordance between studies, including agreement between different tissue types, different microarray platforms, and between neurotoxic and genetic disease models and human Parkinson's disease. Concordance over all studies is low, with correlation of only 0.05 between differential gene expression signatures on average, but increases within human patients and studies of the same tissue type, rising to 0.38 for studies of human substantia nigra. Agreement of animal models, however, is dependent on model type. Studies of brain tissue from Parkinson's disease patients (specifically the substantia nigra) form a distinct group, showing patterns of differential gene expression noticeably different from that in non-brain tissues and animal models of Parkinson's disease; while comparison with other brain diseases (Alzheimer's disease and brain cancer) suggests that the mixed study types display a general signal of neurodegenerative disease. A meta-analysis of these 33 microarray studies demonstrates the greater ability of studies in humans and highly-affected tissues to identify genes previously known to be associated with Parkinson's disease. The observed clustering and concordance results suggest the existence of a 'characteristic' signal of Parkinson's disease found in significantly affected human tissues in humans. These results help to account for the consistency (or lack thereof) so far observed in microarray studies of Parkinson's disease, and act as a guide to the selection of

  3. Use of archived biopsy specimens to study gene expression in oral mucosa from chemotherapy-treated cancer patients.

    Science.gov (United States)

    Mougeot, Jean-Luc C; Mougeot, Farah K B; Peterson, Douglas E; Padilla, Ricardo J; Brennan, Michael T; Lockhart, Peter B

    2013-05-01

    Oral mucositis caused by cancer chemotherapy can result in significant clinical complications. There is a strategic need to accelerate the delineation of the pathobiology. This proof-of-principle study was designed to demonstrate the feasibility of studying archived oral mucosal specimens to further delineate oral mucositis pathobiology. Twenty-nine formalin-fixed and paraffin-embedded tissue blocks of 25-year-old oral mucosa autopsy specimens from cancer chemotherapy patients were studied. Standardized technology was utilized, including RNA isolation and amplification, array hybridization, and gene expression analysis. A predominance of DNA damage in buccal mucosal basal keratinocytes was observed. Data comparing basal cells from buccal vs. gingival mucosa identified differential gene expression of host responses in relation to pathways relevant to oral mucositis pathogenesis, including responses to cancer-associated inflammation. This proof-of-principle study demonstrated that archived oral mucosal specimens may be a potentially valuable resource for the study of oral mucositis in cancer patients. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Studying the effects of genistein on gene expression of fish embryos as an alternative testing approach for endocrine disruption.

    Science.gov (United States)

    Schiller, Viktoria; Wichmann, Arne; Kriehuber, Ralf; Muth-Köhne, Elke; Giesy, John P; Hecker, Markus; Fenske, Martina

    2013-01-01

    Assessment of endocrine disruption currently relies on testing strategies involving adult vertebrates. In order to minimize the use of animal tests according to the 3Rs principle of replacement, reduction and refinement, we propose a transcriptomics and fish embryo based approach as an alternative to identify and analyze an estrogenic activity of environmental chemicals. For this purpose, the suitability of 48 h and 7 days post-fertilization zebrafish and medaka embryos to test for estrogenic disruption was evaluated. The embryos were exposed to the phytoestrogen genistein and subsequently analyzed by microarrays and quantitative real-time PCR. The functional analysis showed that the genes affected related to multiple metabolic and signaling pathways in the early fish embryo, which reflect the known components of genistein's mode of actions, like apoptosis, estrogenic response, hox gene expression and steroid hormone synthesis. Moreover, the transcriptomic data also suggested a thyroidal mode of action and disruption of the nervous system development. The parallel testing of two fish species provided complementary data on the effects of genistein at gene expression level and facilitated the separation of common from species-dependent effects. Overall, the study demonstrated that combining fish embryo testing with transcriptomics can deliver abundant information about the mechanistic effects of endocrine disrupting chemicals, rendering this strategy a promising alternative approach to test for endocrine disruption in a whole organism in-vitro scale system. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Gene expression analysis of 4 biomarker candidates in Eisenia fetida exposed to an environmental metallic trace elements gradient: A microcosm study

    Energy Technology Data Exchange (ETDEWEB)

    Brulle, Franck; Lemiere, Sebastien [Univ Lille Nord de France, F-59000 Lille (France); LGCgE, Equipe Ecologie Numerique et Ecotoxicologie, Lille 1, F-59650 Villeneuve d' Ascq (France); Waterlot, Christophe; Douay, Francis [Univ Lille Nord de France, F-59000 Lille (France); LGCgE, Equipe Sols et Environnement, Groupe ISA, 48 boulevard Vauban, F-59046 Lille Cedex (France); Vandenbulcke, Franck, E-mail: franck.vandenbulcke@univ-lille1.fr [Univ Lille Nord de France, F-59000 Lille (France); LGCgE, Equipe Ecologie Numerique et Ecotoxicologie, Lille 1, F-59650 Villeneuve d' Ascq (France)

    2011-11-15

    Past activities of 2 smelters (Metaleurop Nord and Nyrstar) led to the accumulation of high amounts of Metal Trace Elements (TEs) in top soils of the Noyelles-Godault/Auby area, Northern France. Earthworms were exposed to polluted soils collected in this area to study and better understand the physiological changes, the mechanisms of acclimation, and detoxification resulting from TE exposure. Previously we have cloned and transcriptionally characterized potential biomarkers from immune cells of the ecotoxicologically important earthworm species Eisenia fetida exposed in vivo to TE-spiked standard soils. In the present study, analysis of expression kinetics of four candidate indicator genes (Cadmium-metallothionein, coactosin like protein, phytochelatin synthase and lysenin) was performed in E. fetida after microcosm exposures to natural soils exhibiting an environmental cadmium (Cd) gradient in a kinetic manner. TE body burdens were also measured. This microcosm study provided insights into: (1) the ability of the 4 tested genes to serve as expression biomarkers, (2) detoxification processes through the expression analysis of selected genes, and (3) influence of land uses on the response of potential biomarkers (gene expression or TE uptake). - Highlights: {yields} Expression biomarkers in animals exposed to Cadmium-contaminated field soils. {yields} Expression kinetics to test the ability of genes to serve as expression biomarkers. {yields} Study of detoxification processes through the expression analysis of selected genes.

  6. Glutathione S-transferase omega genes in Alzheimer and Parkinson disease risk, age-at-diagnosis and brain gene expression: an association study with mechanistic implications.

    Science.gov (United States)

    Allen, Mariet; Zou, Fanggeng; Chai, High Seng; Younkin, Curtis S; Miles, Richard; Nair, Asha A; Crook, Julia E; Pankratz, V Shane; Carrasquillo, Minerva M; Rowley, Christopher N; Nguyen, Thuy; Ma, Li; Malphrus, Kimberly G; Bisceglio, Gina; Ortolaza, Alexandra I; Palusak, Ryan; Middha, Sumit; Maharjan, Sooraj; Georgescu, Constantin; Schultz, Debra; Rakhshan, Fariborz; Kolbert, Christopher P; Jen, Jin; Sando, Sigrid B; Aasly, Jan O; Barcikowska, Maria; Uitti, Ryan J; Wszolek, Zbigniew K; Ross, Owen A; Petersen, Ronald C; Graff-Radford, Neill R; Dickson, Dennis W; Younkin, Steven G; Ertekin-Taner, Nilüfer

    2012-04-11

    Glutathione S-transferase omega-1 and 2 genes (GSTO1, GSTO2), residing within an Alzheimer and Parkinson disease (AD and PD) linkage region, have diverse functions including mitigation of oxidative stress and may underlie the pathophysiology of both diseases. GSTO polymorphisms were previously reported to associate with risk and age-at-onset of these diseases, although inconsistent follow-up study designs make interpretation of results difficult. We assessed two previously reported SNPs, GSTO1 rs4925 and GSTO2 rs156697, in AD (3,493 ADs vs. 4,617 controls) and PD (678 PDs vs. 712 controls) for association with disease risk (case-controls), age-at-diagnosis (cases) and brain gene expression levels (autopsied subjects). We found that rs156697 minor allele associates with significantly increased risk (odds ratio = 1.14, p = 0.038) in the older ADs with age-at-diagnosis > 80 years. The minor allele of GSTO1 rs4925 associates with decreased risk in familial PD (odds ratio = 0.78, p = 0.034). There was no other association with disease risk or age-at-diagnosis. The minor alleles of both GSTO SNPs associate with lower brain levels of GSTO2 (p = 4.7 × 10-11-1.9 × 10-27), but not GSTO1. Pathway analysis of significant genes in our brain expression GWAS, identified significant enrichment for glutathione metabolism genes (p = 0.003). These results suggest that GSTO locus variants may lower brain GSTO2 levels and consequently confer AD risk in older age. Other glutathione metabolism genes should be assessed for their effects on AD and other chronic, neurologic diseases.

  7. Noise minimization in eukaryotic gene expression.

    Directory of Open Access Journals (Sweden)

    Hunter B Fraser

    2004-06-01

    Full Text Available All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or "noise." Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  8. Noise minimization in eukaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-15

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  9. Paternally expressed genes predominate in the placenta.

    Science.gov (United States)

    Wang, Xu; Miller, Donald C; Harman, Rebecca; Antczak, Douglas F; Clark, Andrew G

    2013-06-25

    The discovery of genomic imprinting through studies of manipulated mouse embryos indicated that the paternal genome has a major influence on placental development. However, previous research has not demonstrated paternal bias in imprinted genes. We applied RNA sequencing to trophoblast tissue from reciprocal hybrids of horse and donkey, where genotypic differences allowed parent-of-origin identification of most expressed genes. Using this approach, we identified a core group of 15 ancient imprinted genes, of which 10 were paternally expressed. An additional 78 candidate imprinted genes identified by RNA sequencing also showed paternal bias. Pyrosequencing was used to confirm the imprinting status of six of the genes, including the insulin receptor (INSR), which may play a role in growth regulation with its reciprocally imprinted ligand, histone acetyltransferase-1 (HAT1), a gene involved in chromatin modification, and lymphocyte antigen 6 complex, locus G6C, a newly identified imprinted gene in the major histocompatibility complex. The 78 candidate imprinted genes displayed parent-of-origin expression bias in placenta but not fetus, and most showed less than 100% silencing of the imprinted allele. Some displayed variability in imprinting status among individuals. This variability results in a unique epigenetic signature for each placenta that contributes to variation in the intrauterine environment and thus presents the opportunity for natural selection to operate on parent-of-origin differential regulation. Taken together, these features highlight the plasticity of imprinting in mammals and the central importance of the placenta as a target tissue for genomic imprinting.

  10. Detection of gene expression pattern in the early stage after spinal cord injury by gene chip

    Institute of Scientific and Technical Information of China (English)

    刘成龙; 靳安民; 童斌辉

    2003-01-01

    Objective: To study the changes of the gene expression pattern of spinal cord tissues in the early stage after injury by DNA microarray (gene chip). Methods: The contusion model of rat spinal cord was established according to Allen's falling strike method and the gene expression patterns of normal and injured spinal cord tissues were studied by gene chip. Results: The expression of 45 genes was significantly changed in the early stage after spinal cord injury, in which 22 genes up-regulated and 23 genes down-regulated. Conclusions: The expression of some genes changes significantly in the early stage after spinal cord injury, which indicates the complexity of secondary spinal cord injury.

  11. Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

    Directory of Open Access Journals (Sweden)

    Kathleen D Cusick

    Full Text Available Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1 light/dark cycling: btl, asl, and vma1; (2 all-dark growth: btl, tbp, vma1, and vma2; (3 temperature flux: btl, vma1, act, and asl; (4 all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated, expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring

  12. Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

    Science.gov (United States)

    Cusick, Kathleen D; Fitzgerald, Lisa A; Pirlo, Russell K; Cockrell, Allison L; Petersen, Emily R; Biffinger, Justin C

    2014-01-01

    Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene

  13. YKL-40 Gene Expression and Plasma Levels of CD30 are not Affected by Isoflurane or Propofol: Pilot Study

    Directory of Open Access Journals (Sweden)

    Shirin Lak

    2016-10-01

    Full Text Available Background: It has been hypothesized that the body's response to anesthesia techniques can increase risk of cancer recurrence and metastatic disease after surgery and also can modulate immune responses. Some acute inflammatory markers have been measured to survey the immunomodulatory effect of anesthesia, but in this research, we studied the plasma level of CD30 and YKL-40 gene expression which can present major changes of the immune system.Materials and Methods: Our study was a controlled before and after study. 34 women with biopsy-proven breast cancer were randomized to receive either propofol general anesthesia (n=17 or standard isoflurane general anesthesia (n=17. There were no significant differences between the two patient groups in age, body weight, and height, length of general anesthesia, operative time and group of surgery. The blood samples were collected in two different sets, before anesthesia and 72-h postoperatively. Soluble CD30 (sCD30 plasma level was measured by ELISA and YKL-40/CHI3L1 gene expression was evaluated by real-time-PCR.Results: The results showed that the anesthetics, propofol and isoflurane, have no effect on the expression of YKL-40. Despite increased in the expression of YKL-40 that was observed in patients receiving isoflurane, this increase was not statistically significant. There was no significant increase or decrease in plasma concentrations of sCD30.Conclusion: YKL-40 and sCD30 are not affected by isoflurane or propofol.  So, in immunological perspective, there is no preference in use of isoflurane or propofol in breast cancer patients.

  14. Use of somatic cells from goat milk for dynamic studies of gene expression in the mammary gland.

    Science.gov (United States)

    Boutinaud, M; Rulquin, H; Keisler, D H; Djiane, J; Jammes, H

    2002-05-01

    Somatic cells are present in the milk throughout lactation and consist of leukocytes and epithelial cells exfoliated from the mammary epithelium. Our objective was to determine the efficacy of using somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. Over a 4-wk interval, cells were isolated from daily morning milk samples and samples taken 30 min after milking. They were characterized by direct cell counts and by flow cytometry analysis after immunostaining with antibodies directed against cytokeratin and CD45, a common leukocyte antigen. Epithelial cell counts within the morning milk ranged from 15 to 45% of total milk somatic cells. After-milking samples contained twice as many cells as did morning milk samples. The RNA was extracted from the somatic cells of both types of milk samples with equivalent efficiency (a mean of 1.2 microg RNA/mL of milk). Four mRNA variants of the alpha-S1 casein gene were detected by Northern blot analysis and the amount of each mRNA in milk cells was related to protein concentration in milk. The comparison between mRNA from the mammary gland and from congruently collected milk cells showed that relative amounts of mRNA for each milk-protein (alpha-S1 and kappa-casein and alactalbumin) were conserved. In a third experiment, daily milk cell RNA preparations were extracted to assess the effect of growth hormone (GH) on mammary gene expression; four goats were separated into two groups in order to perform a switch-back design consisting of three treatment weeks: Control, GH-Control or GH-Control-GH. In this study, treatment of goats with GH increased milk yields by 5%. Throughout the control and GH treatments, the expression of the three milk-protein genes studied were highly and significantly correlated (r = 0.949 and r = 0.958, P milk-protein mRNA abundances increased with the same pattern. In conclusion, the opportunity to use milk somatic cells for RNA preparation and analysis provides a

  15. Classification with binary gene expressions

    OpenAIRE

    Tuna, Salih; Niranjan, Mahesan

    2009-01-01

    Microarray gene expression measurements are reported, used and archived usually to high numerical precision. However, properties of mRNA molecules, such as their low stability and availability in small copy numbers, and the fact that measurements correspond to a population of cells, rather than a single cell, makes high precision meaningless. Recent work shows that reducing measurement precision leads to very little loss of information, right down to binary levels. In this paper we show how p...

  16. Early gene expression changes with rush immunotherapy

    Directory of Open Access Journals (Sweden)

    Barnett Sherry

    2011-09-01

    Full Text Available Abstract Background To examine whether whole genome expression profiling could reveal changes in mRNA expression of peripheral blood mononuclear cells (PBMC from allergic patients undergoing rush immunotherapy (RIT that might be manifest within the first few months of treatment. Methods For this study, PBMC from three allergic patients undergoing RIT were assessed at four timepoints: prior to RIT, at 1 week and 7 week post-RIT, during build-up and at 4 months, after establishment of a maintenance dose. PBMC mRNA gene expression changes over time were determined by oligonucleotide microarrays using the Illumina Human-6 BeadChip Platform, which simultaneously interrogates expression profiles of > 47,000 transcripts. Differentially expressed genes were identified using well-established statistical analysis for microarrays. In addition, we analyzed peripheral blood basophil high-affinity IgE receptor (Fc epsilon RI expression and T-regulatory cell frequency as detected by expression of CD3+CD4+CD25bright cells at each timepoint using flow cytometry. Results In comparing the initial 2 timepoints with the final 2 timepoints and analyzing for genes with ≥1.5-fold expression change (p less than or equal to 0.05, BH-FDR, we identified 507 transcripts. At a 2-fold change (p less than or equal to 0.05, BH-FDR, we found 44 transcripts. Of these, 28 were up-regulated and 16 were down-regulated genes. From these datasets, we have identified changes in immunologically relevant genes from both the innate and adaptive response with upregulation of expressed genes for molecules including IL-1β, IL-8, CD40L, BTK and BCL6. At the 4 month timepoint, we noted a downward trend in Fc epsilon RI expression in each of the three patients and increased allergen-specific IgG4 levels. No change was seen in the frequency of peripheral T-regulatory cells expressed over the four timepoints. Conclusions We observed significant changes in gene expression early in peripheral

  17. Expression of Helicobacter pylori hspA Gene in Lactococcus lactis NICE System and Experimental Study on Its Immunoreactivity

    Directory of Open Access Journals (Sweden)

    Xiao-Juan Zhang

    2015-01-01

    Full Text Available Aim. The aim of this study was to develop an oral Lactococcus lactis (L. lactis vaccine against Helicobacter pylori (H. pylori. Methods. After L. lactis NZ3900/pNZ8110-hspA was constructed, growth curves were plotted to study whether the growth of recombinant L. lactis was affected after hspA was cloned into L. lactis and whether the growth of empty bacteria, empty plasmid bacteria, and recombinant L. lactis was affected by different concentrations of Nisin; SDS-PAGE and Western blot were adopted, respectively, to detect the HspA expressed by recombinant L. lactis and its immunoreactivity. Results. There was no effect observed from the growth curve after exogenous gene hspA was cloned into L. lactis NZ3900; different concentrations of Nisin did not affect the growth of NZ3900 and NZ3900/pNZ8110, while different concentrations of Nisin inhibited the growth of NZ3900/pNZ8110-hspA except 10 ng/mL Nisin. No HspA strip was observed from SDS-PAGE. Western blot analysis showed that HspA expressed by recombinant bacteria had favorable immunoreactivity. Conclusion. The growth of recombinant L. lactis was suppressed even though a small amount of HspA had been induced to express. Therefore recombinant L. lactis only express HspA which was not suitable to be oral vaccine against Helicobacter pylori.

  18. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

    Directory of Open Access Journals (Sweden)

    Lin-Lin Liu

    Full Text Available Reverse transcription-quantitative polymerase chain reaction (RT-qPCR is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct, and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2 expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

  19. Progress in the Study of Acupuncture in Regulating Post-Cerebral Ischemia/Reperfusion Cell-Apoptosis Related Gene Expression

    Institute of Scientific and Technical Information of China (English)

    卜渊; 耿德勤; 曾因明

    2003-01-01

    @@ Cerebralvascular disease has already become one of the serious illnesses that threatens human health. Along with the development of medicine, although the therapeutic method harvested huge progress, currently ideal therapeutic methods are lacking. The conventional acupuncture has definite therapeutic effect on cerebropathy. Clinical practice and various animal experiments confirmed that acupuncture could alleviate the pathologic damage after cerebral ischemic injury and promote the nerve function recovery. Past studies showed that the role of acupuncture in treating cerebral ischemia is realized through alleviating post-ischemic neuron necrosis, while recent study discovered that acupuncture has inhibitory effect on post-ischemia induced neuronal necrosis(1), which brought the mechanism of acupuncture in treating cerebral ischemia from the biochemical and metabolical level to the molecular biologic level. The studies revealed that after cerebral ischemia, many genes were induced to express themselves, protein product they coded directly or indirectly participated in the regulation of post-cerebral ischemia apoptosis of neuron, some promoting the apoptosis, while others inhibiting apoptosis with some of the function still unclear. The anti-apoptotic effect of acupuncture is accomplished through regulating the relevant apoptotic gene expression(2), and now it is reviewed as follows:

  20. Genome-wide annotation, expression profiling, and protein interaction studies of the core cell-cycle genes in Phalaenopsis aphrodite.

    Science.gov (United States)

    Lin, Hsiang-Yin; Chen, Jhun-Chen; Wei, Miao-Ju; Lien, Yi-Chen; Li, Huang-Hsien; Ko, Swee-Suak; Liu, Zin-Huang; Fang, Su-Chiung

    2014-01-01

    Orchidaceae is one of the most abundant and diverse families in the plant kingdom and its unique developmental patterns have drawn the attention of many evolutionary biologists. Particular areas of interest have included the co-evolution of pollinators and distinct floral structures, and symbiotic relationships with mycorrhizal flora. However, comprehensive studies to decipher the molecular basis of growth and development in orchids remain scarce. Cell proliferation governed by cell-cycle regulation is fundamental to growth and development of the plant body. We took advantage of recently released transcriptome information to systematically isolate and annotate the core cell-cycle regulators in the moth orchid Phalaenopsis aphrodite. Our data verified that Phalaenopsis cyclin-dependent kinase A (CDKA) is an evolutionarily conserved CDK. Expression profiling studies suggested that core cell-cycle genes functioning during the G1/S, S, and G2/M stages were preferentially enriched in the meristematic tissues that have high proliferation activity. In addition, subcellular localization and pairwise interaction analyses of various combinations of CDKs and cyclins, and of E2 promoter-binding factors and dimerization partners confirmed interactions of the functional units. Furthermore, our data showed that expression of the core cell-cycle genes was coordinately regulated during pollination-induced reproductive development. The data obtained establish a fundamental framework for study of the cell-cycle machinery in Phalaenopsis orchids.

  1. Differential expression of oxidative stress and inflammation related genes in peripheral blood mononuclear cells in response to a low-calorie diet: a nutrigenomics study.

    Science.gov (United States)

    Crujeiras, Ana B; Parra, Dolores; Milagro, Fermín I; Goyenechea, Estibaliz; Larrarte, Eider; Margareto, Javier; Martínez, J Alfredo

    2008-12-01

    Nutrigenomics is a new application of omics technologies in nutritional science. Nutrigenomics aims to identify molecular markers of diet-related diseases and mechanisms of interindividual variability in response to food. The aim of this study was to evaluate peripheral blood mononuclear cells (PBMC) as a model system and readily available source of RNA to discern gene expression signatures in relation to personalized therapy of obesity. PBMC were collected from obese men before and after an 8-week low-calorie diet (LCD) to lose weight. Changes in gene expression before and after the LCD were initially screened using a DNA-microarray platform and validated by qRT-PCR. Global gene expression analysis identified 385 differentially expressed transcripts after the LCD. Further analyses showed a decrease in some specific oxidative stress and inflammation genes. Interestingly, expression of these genes was directly related to body weight, while a lower IL8 gene expression was associated with higher fat mass decrease. Collectively, these observations suggest that PBMCs are a suitable RNA source and model system to perform nutrigenomics studies related to obesity and development of personalized dietary treatments. IL8 gene expression warrant further research as a putative novel biomarker of changes in body fat percentage in response to an LCD.

  2. X chromosome regulation of autosomal gene expression in bovine blastocysts

    Science.gov (United States)

    Itoh, Yuichiro; Arnold, Arthur P.

    2014-01-01

    Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male to female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient. PMID:24817096

  3. Mining the tissue-tissue gene co-expression network for tumor microenvironment study and biomarker prediction

    OpenAIRE

    2013-01-01

    Background Recent discovery in tumor development indicates that the tumor microenvironment (mostly stroma cells) plays an important role in cancer development. To understand how the tumor microenvironment (TME) interacts with the tumor, we explore the correlation of the gene expressions between tumor and stroma. The tumor and stroma gene expression data are modeled as a weighted bipartite network (tumor-stroma coexpression network) where the weight of an edge indicates the correlation between...

  4. Gene Expression in the Human Endolymphatic Sac

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

    2015-01-01

    OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the solute carrier (SLC) molecules of ion transporters in the human endolymphatic sac. STUDY DESIGN: cDNA microarrays and immunohistochemistry were used for analyses...... of fresh human endolymphatic sac tissue samples. METHODS: Twelve tissue samples of the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample expression of solute carrier family genes, using adjacent dura...... mater as control. Immunohistochemistry was used for verification of translation of selected genes, as well as localization of the specific protein within the sac. RESULTS: An extensive representation of the SLC family genes were upregulated in the human endolymphatic sac, including SLC26a4 Pendrin, SLC4...

  5. Expression studies and promoter analysis of the nuclear gene for mitochondrial transcription factor 1 (MTF1) in yeast.

    Science.gov (United States)

    Jan, P S; Stein, T; Hehl, S; Lisowsky, T

    1999-08-01

    The basal mitochondrial transcription apparatus of Saccharomyces cerevisiae consists of the core enzyme for mitochondrial RNA polymerase and the specificity factor. The core enzyme is homologous to those of bacteriophages T3, T7 and SP6 whereas the specificity factor shows similarities with bacterial sigma factors. Recently it was shown that the bacteriophage-type core enzyme is widespread among the eukaryotic lineage and a common picture for the mitochondrial transcription apparatus in eukaryotic cells is now emerging. In contrast to the situation for the core enzyme, the gene for the specificity factor has only been identified from S. cerevisiae and more recently from two other yeast species. As the specificity factor is the key component for initiation of transcription at the mitochondrial promoter we wanted to study in more detail gene expression, regulation, and the function of the promoter of the nuclear MTF1 gene. For this purpose the messenger RNA level for scMTF1 was investigated under a large number of different growth conditions and thereby exhibited a very low, but regulated and carbon source-dependent, expression. Deletion experiments identify the minimal promoter for functional complementation in yeast. To evaluate the functional conservation of the promoter elements the homologous MTF1 gene from the closely related yeast Saccharomyces douglasii was isolated and tested in heterologous complementation experiments. In spite of a highly conserved protein sequence these studies demonstrate that at low-copy number sdMTF1 is not able to substitute for scMTF1 in S. cerevisiae. Promoter exchange experiments with MTF1 from S. cerevisiae and S. douglasii demonstrate that differences in gene expression are responsible for the failure in heterologous complementation. This finding prompted us to compare the promoter regions of MTF1 from four different yeast species. For this purpose the sequences of the 5' regions from S. douglasii, S. kluyveri and Kluyveromyces

  6. Identification and evaluation of reference genes for expression studies by RT-qPCR during embryonic development of the emerging model organism, Macrobrachium olfersii.

    Science.gov (United States)

    Jaramillo, Michael L; Ammar, Dib; Quispe, Ruth L; Guzman, Frank; Margis, Rogerio; Nazari, Evelise M; Müller, Yara M R

    2017-01-20

    RT-qPCR is a sensitive and highly efficient technique that is widely used in gene expression analysis and to provide insight into the molecular mechanisms underlying embryonic development. The freshwater prawn, Macrobrachium olfersii is an emerging model organism, but, the stable reference genes of this species need to be identified and validated for RT-qPCR analysis. Thus, the aim of this study was to evaluate the expression stability of six genes (β-act, GAPDH, EF-1α, RpL8, RpS6, AK) in embryos and in adult tissues (cerebral ganglia, muscle and hepatopancreas) of M. olfersii. The expression stabilities of these genes were evaluated using geNorm, NormFinder, BestKeeper, ΔCt method and integrated tool RefFinder. In the general ranking, RpL8 and RpS6 were the most stable genes in embryos, while RpS6 and RpL8 were the most stable in a combined adult tissue analysis. Analysis of the adult tissues revealed that β-act and AK were the most stable genes in cerebral ganglia, RpL8 and AK in muscle, and RpS6 and β-act in hepatopancreas. EF-1α and GAPDH were the least stable genes and as normalizer genes in RT-qPCR affected expression of the Distal-less gene during M. olfersii development. This study provides suitable reference genes for RT-qPCR analysis and allows future studies of the gene expression in M. olfersii for understanding the molecular mechanisms of their development. To our knowledge, this is the first published study that identifies and evaluates reference genes for RT-qPCR analysis in M. olfersii and could be useful as basis for evaluations of reference genes in other prawns.

  7. Integrating pathway analysis and genetics of gene expression for genome-wide association study of basal cell carcinoma.

    Science.gov (United States)

    Zhang, Mingfeng; Liang, Liming; Morar, Nilesh; Dixon, Anna L; Lathrop, G Mark; Ding, Jun; Moffatt, Miriam F; Cookson, William O C; Kraft, Peter; Qureshi, Abrar A; Han, Jiali

    2012-04-01

    Genome-wide association studies (GWASs) have primarily focused on marginal effects for individual markers and have incorporated external functional information only after identifying robust statistical associations. We applied a new approach combining the genetics of gene expression and functional classification of genes to the GWAS of basal cell carcinoma (BCC) to identify potential biological pathways associated with BCC. We first identified 322,324 expression-associated single-nucleotide polymorphisms (eSNPs) from two existing GWASs of global gene expression in lymphoblastoid cell lines (n = 955), and evaluated the association of these functionally annotated SNPs with BCC among 2,045 BCC cases and 6,013 controls in Caucasians. We then grouped them into 99 KEGG pathways for pathway analysis and identified two pathways associated with BCC with p value <0.05 and false discovery rate (FDR) <0.5: the autoimmune thyroid disease pathway (mainly HLA class I and II antigens, p < 0.001, FDR = 0.24) and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway (p = 0.02, FDR = 0.49). Seventy-nine (25.7%) out of 307 significant eSNPs in the JAK-STAT pathway were associated with BCC risk (p < 0.05) in an independent replication set of 278 BCC cases and 1,262 controls. In addition, the association of JAK-STAT signaling pathway was marginally validated using 16,691 eSNPs identified from 110 normal skin samples (p = 0.08). Based on the evidence of biological functions of the JAK-STAT pathway on oncogenesis, it is plausible that this pathway is involved in BCC pathogenesis.

  8. Molecular study of the chlorella algae and determining its functional features with the approach of hydrogenase gene expression

    Directory of Open Access Journals (Sweden)

    Seyedeh Tayebeh Mousavi

    2016-09-01

    Full Text Available Biohydrogen production by biological processes are known as a renewable energy source. The aim of the investigation was to find and optimize the most appropriate medium for the algae growth to produce the maximum amount of hydrogen. First of all, the bioinformatics and biosystematics studies were taken for identifying the collected microalgae which was detected as Chlorella with the following features: spherical appearance, spent protozoan, Cup-shaped chloroplast with no flagella. On the other hand, the molecular analysis by PCR and 18S sequence typing of interested microalgae demonstrated 100% similarity to that well known sequences for Chlorella vulgaris. Second, we assessed some culture media including BBM, Chu10, TAP, and Sorokin and Krauss for optimum growth conditions for Chlorella vulgaris. In general, our results showed that BBM medium had the highest efficiency for producing microalgae biomass under following conditions: pH=8, temperature of 30 ° with 16 to 8 h light to darkness periods ratio.Third, we designed a more efficient photo-bioreactor apparatus toward inducing more powerful bio-hydrogen production by hydrogenase enzyme activity of our given microalgae. Then, the performance of the apparatus as well as the gene expression was scrutinized under different conditions (light, pH, sulphorous, etc.. For this, after extracting RNA and constructing cDNA, hydrogenase gene was amplified with PCR and the product was evaluated by agarose gel. However, the relative expression of the gene measured by Real Time PCR showed the influence of light, pH and sulphourous on the expression as compared with control.

  9. Expression studies of six human obesity-related genes in seven tissues from divergent pig breeds

    DEFF Research Database (Denmark)

    Cirera, S.; Jensen, M. S.; Elbrønd, V. S.;

    2014-01-01

    receptor (MC4R), fat mass and obesity associated (FTO), neuronal growth regulator 1 (NEGR)1 and adiponectin (ADIPOQ), in seven obesity-relevant tissues (liver; muscle; pancreas; hypothalamus; and retroperitoneal, subcutaneous and mesenteric adipose tissues) in two pig breeds (production pigs and Göttingen...... minipigs) that deviate phenotypically and genetically from each other with respect to obesity traits. We observe significant differential expression for LEP, LEPR and ADIPOQ in muscle and in all three adipose tissues. Interestingly, in pancreas, LEP expression is only detected in the fat minipigs. FTO...... shows significant differential expression in all tissues analyzed, and NEGR1 shows significant differential expression in muscle, pancreas, hypothalamus and subcutaneous adipose tissue. The MC4R transcript can be detected only in hypothalamus. In general, the expression profiles of the investigated...

  10. Gene expression profiling of human erythroid progenitors by micro-serial analysis of gene expression.

    Science.gov (United States)

    Fujishima, Naohito; Hirokawa, Makoto; Aiba, Namiko; Ichikawa, Yoshikazu; Fujishima, Masumi; Komatsuda, Atsushi; Suzuki, Yoshiko; Kawabata, Yoshinari; Miura, Ikuo; Sawada, Ken-ichi

    2004-10-01

    We compared the expression profiles of highly purified human CD34+ cells and erythroid progenitor cells by micro-serial analysis of gene expression (microSAGE). Human CD34+ cells were purified from granulocyte colony-stimulating factor-mobilized blood stem cells, and erythroid progenitors were obtained by cultivating these cells in the presence of stem cell factor, interleukin 3, and erythropoietin. Our 10,202 SAGE tags allowed us to identify 1354 different transcripts appearing more than once. Erythroid progenitor cells showed increased expression of LRBA, EEF1A1, HSPCA, PILRB, RANBP1, NACA, and SMURF. Overexpression of HSPCA was confirmed by real-time polymerase chain reaction analysis. MicroSAGE revealed an unexpected preferential expression of several genes in erythroid progenitor cells in addition to the known functional genes, including hemoglobins. Our results provide reference data for future studies of gene expression in various hematopoietic disorders, including myelodysplastic syndrome and leukemia.

  11. Gene expression profiles predictive of outcome and age in infant acute lymphoblastic leukemia: A Children's Oncology Group study

    NARCIS (Netherlands)

    H. Kang; C.S. Wilson (Carla); R. Harvey (R.); I.-M. Chen (I.-Ming); M.H. Murphy (Maurice); S.R. Atlas (Susan); E.J. Bedrick (Edward); M. Devidas (Meenakshi); A.J. Carroll; B.W. Robinson (Blaine); R.W. Stam (Ronald); M.G. Valsecchi (Maria Grazia); R. Pieters (Rob); N.A. Heerema (Nyla); J.M. Hilden (Joanne); C.A. Felix (Carolyn); G.H. Reaman (Gregory); B. Camitta (Bruce); N.J. Winick (Naomi); W.L. Carroll (William); S.D. Dreyer; S.P. Hunger (Stephen); S.F. Willman (Sami )

    2012-01-01

    textabstractGene expression profiling was performed on 97 cases of infant ALL from Children's Oncology Group Trial P9407. Statistical modeling of an outcome predictor revealed 3 genes highly predictive of event-free survival (EFS), beyond age and MLL status: FLT3, IRX2, and TACC2. Low FLT3 expressio

  12. Hormone-replacement therapy influences gene expression profiles and is associated with breast-cancer prognosis: a cohort study

    Directory of Open Access Journals (Sweden)

    Skoog Lambert

    2006-06-01

    Full Text Available Abstract Background Postmenopausal hormone-replacement therapy (HRT increases breast-cancer risk. The influence of HRT on the biology of the primary tumor, however, is not well understood. Methods We obtained breast-cancer gene expression profiles using Affymetrix human genome U133A arrays. We examined the relationship between HRT-regulated gene profiles, tumor characteristics, and recurrence-free survival in 72 postmenopausal women. Results HRT use in patients with estrogen receptor (ER protein positive tumors (n = 72 was associated with an altered regulation of 276 genes. Expression profiles based on these genes clustered ER-positive tumors into two molecular subclasses, one of which was associated with HRT use and had significantly better recurrence free survival despite lower ER levels. A comparison with external data suggested that gene regulation in tumors associated with HRT was negatively correlated with gene regulation induced by short-term estrogen exposure, but positively correlated with the effect of tamoxifen. Conclusion Our findings suggest that post-menopausal HRT use is associated with a distinct gene expression profile related to better recurrence-free survival and lower ER protein levels. Tentatively, HRT-associated gene expression in tumors resembles the effect of tamoxifen exposure on MCF-7 cells.

  13. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

  14. Generalised Anxiety Disorder--A Twin Study of Genetic Architecture, Genome-Wide Association and Differential Gene Expression.

    Directory of Open Access Journals (Sweden)

    Matthew N Davies

    Full Text Available Generalised Anxiety Disorder (GAD is a common anxiety-related diagnosis, affecting approximately 5% of the adult population. One characteristic of GAD is a high degree of anxiety sensitivity (AS, a personality trait which describes the fear of arousal-related sensations. Here we present a genome-wide association study of AS using a cohort of 730 MZ and DZ female twins. The GWAS showed a significant association for a variant within the RBFOX1 gene. A heritability analysis of the same cohort also confirmed a significant genetic component with h2 of 0.42. Additionally, a subset of the cohort (25 MZ twins discordant for AS was studied for evidence of differential expression using RNA-seq data. Significant differential expression of two exons with the ITM2B gene within the discordant MZ subset was observed, a finding that was replicated in an independent cohort. While previous research has shown that anxiety has a high comorbidity with a variety of psychiatric and neurodegenerative disorders, our analysis suggests a novel etiology specific to AS.

  15. Generalised Anxiety Disorder--A Twin Study of Genetic Architecture, Genome-Wide Association and Differential Gene Expression.

    Science.gov (United States)

    Davies, Matthew N; Verdi, Serena; Burri, Andrea; Trzaskowski, Maciej; Lee, Minyoung; Hettema, John M; Jansen, Rick; Boomsma, Dorret I; Spector, Tim D

    2015-01-01

    Generalised Anxiety Disorder (GAD) is a common anxiety-related diagnosis, affecting approximately 5% of the adult population. One characteristic of GAD is a high degree of anxiety sensitivity (AS), a personality trait which describes the fear of arousal-related sensations. Here we present a genome-wide association study of AS using a cohort of 730 MZ and DZ female twins. The GWAS showed a significant association for a variant within the RBFOX1 gene. A heritability analysis of the same cohort also confirmed a significant genetic component with h2 of 0.42. Additionally, a subset of the cohort (25 MZ twins discordant for AS) was studied for evidence of differential expression using RNA-seq data. Significant differential expression of two exons with the ITM2B gene within the discordant MZ subset was observed, a finding that was replicated in an independent cohort. While previous research has shown that anxiety has a high comorbidity with a variety of psychiatric and neurodegenerative disorders, our analysis suggests a novel etiology specific to AS.

  16. Gene expression profiling of the hippocampal dentate gyrus in an adult toxicity study captures a variety of neurodevelopmental dysfunctions in rat models of hypothyroidism.

    Science.gov (United States)

    Shiraki, Ayako; Saito, Fumiyo; Akane, Hirotoshi; Akahori, Yumi; Imatanaka, Nobuya; Itahashi, Megu; Yoshida, Toshinori; Shibutani, Makoto

    2016-01-01

    We previously found that developmental hypothyroidism changed the expression of genes in the rat hippocampal dentate gyrus, a brain region where adult neurogenesis is known to occur. In the present study, we performed brain region-specific global gene expression profiling in an adult rat hypothyroidism model to see if it reflected the developmental neurotoxicity we saw in the developmental hypothyroidism model. Starting when male rats were 5 weeks old, we administered 6-propyl-2-thiouracil at a doses of 0, 0.1 and 10 mg kg(-1) body weight by gavage for 28 days. We selected four brain regions to represent both cerebral and cerebellar tissues: hippocampal dentate gyrus, cerebral cortex, corpus callosum and cerebellar vermis. We observed significant alterations in the expression of genes related to neural development (Eph family genes and Robo3) in the cerebral cortex and hippocampal dentate gyrus and in the expression of genes related to myelination (Plp1 and Mbp) in the hippocampal dentate gyrus. We observed only minor changes in the expression of these genes in the corpus callosum and cerebellar vermis. We used real-time reverse-transcription polymerase chain reaction to confirm Chrdl1, Hes5, Mbp, Plp1, Slit1, Robo3 and the Eph family transcript expression changes. The most significant changes in gene expression were found in the dentate gyrus. Considering that the gene expression profile of the adult dentate gyrus closely related to neurogenesis, 28-day toxicity studies looking at gene expression changes in adult hippocampal dentate gyrus may also detect possible developmental neurotoxic effects.

  17. Microarray Analysis Reveals Higher Gestational Folic Acid Alters Expression of Genes in the Cerebellum of Mice Offspring—A Pilot Study

    Directory of Open Access Journals (Sweden)

    Subit Barua

    2015-01-01

    Full Text Available Folate is a water-soluble vitamin that is critical for nucleotide synthesis and can modulate methylation of DNA by altering one-carbon metabolism. Previous studies have shown that folate status during pregnancy is associated with various congenital defects including the risk of aberrant neural tube closure. Maternal exposure to a methyl supplemented diet also can alter DNA methylation and gene expression, which may influence the phenotype of offspring. We investigated if higher gestational folic acid (FA in the diet dysregulates the expression of genes in the cerebellum of offspring in C57BL/6 J mice. One week before gestation and throughout the pregnancy, groups of dams were supplemented with FA either at 2 mg/kg or 20 mg/kg of diet. Microarray analysis was used to investigate the genome wide gene expression profile in the cerebellum from day old pups. Our results revealed that exposure to the higher dose FA diet during gestation dysregulated expression of several genes in the cerebellum of both male and female pups. Several transcription factors, imprinted genes, neuro-developmental genes and genes associated with autism spectrum disorder exhibited altered expression levels. These findings suggest that higher gestational FA potentially dysregulates gene expression in the offspring brain and such changes may adversely alter fetal programming and overall brain development.

  18. Phytochrome-regulated Gene Expression

    Institute of Scientific and Technical Information of China (English)

    Peter H. Quail

    2007-01-01

    Identification of all genes involved in the phytochrome (phy)-mediated responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth and development. This article highlights and integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome-wide set of phy-regulated genes that respond rapidly to red-light signals upon first exposure of dark-grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an initial subset of these genes. The data: (a) reveal considerable complexity in the channeling of the light signals through the different phy-family members (phyA to phyE) to responsive genes; (b) identify a diversity of transcription-factor-encoding genes as major early, if not primary, targets of phy signaling, and, therefore, as potentially important regulators in the transcriptional-network hierarchy; and (c) identify auxin-related genes as the dominant class among rapidly-regulated, hormone-related genes. However, reverse-genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy-induced seedling deetiolation.

  19. Predicting metastasized seminoma using gene expression.

    Science.gov (United States)

    Ruf, Christian G; Linbecker, Michael; Port, Matthias; Riecke, Armin; Schmelz, Hans U; Wagner, Walter; Meineke, Victor; Abend, Michael

    2012-07-01

    Treatment options for testis cancer depend on the histological subtype as well as on the clinical stage. An accurate staging is essential for correct treatment. The 'golden standard' for staging purposes is CT, but occult metastasis cannot be detected with this method. Currently, parameters such as primary tumour size, vessel invasion or invasion of the rete testis are used for predicting occult metastasis. Last year the association of these parameters with metastasis could not be validated in a new independent cohort. Gene expression analysis in testis cancer allowed discrimination between the different histological subtypes (seminoma and non-seminoma) as well as testis cancer and normal testis tissue. In a two-stage study design we (i) screened the whole genome (using human whole genome microarrays) for candidate genes associated with the metastatic stage in seminoma and (ii) validated and quantified gene expression of our candidate genes (real-time quantitative polymerase chain reaction) on another independent group. Gene expression measurements of two of our candidate genes (dopamine receptor D1 [DRD1] and family with sequence similarity 71, member F2 [FAM71F2]) examined in primary testis cancers made it possible to discriminate the metastasis status in seminoma. The discriminative ability of the genes exceeded the predictive significance of currently used histological/pathological parameters. Based on gene expression analysis the present study provides suggestions for improved individual decision making either in favour of early adjuvant therapy or increased surveillance. To evaluate the usefulness of gene expression profiling for predicting metastatic status in testicular seminoma at the time of first diagnosis compared with established clinical and pathological parameters. Total RNA was isolated from testicular tumours of metastasized patients (12 patients, clinical stage IIa-III), non-metastasized patients (40, clinical stage I) and adjacent 'normal' tissue

  20. Selection of relatively exact reference genes for gene expression studies in flixweed (Descurainia sophia) by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Xu, Xian; Liu, Xiaomin; Chen, Silong; Li, Binghua; Wang, Xiaoyun; Fan, Cuiqin; Wang, Guiqi; Ni, Hanwen

    2016-02-01

    The reliable gene expression analysis of a reverse transcription quantitative polymerase chain reaction (qRT-PCR) mainly depends on selecting suitable reference genes that can express stably under different experimental conditions. Thus far, no reference genes have been identified in flixweed. In this paper, 7 supposed reference genes were selected to evaluate their expression stabilities by qRT-PCR in flixweed under three conditions including different sampling times after tribenuron treatment, different organs, and different growth stages using geNorm, NormFinder, and BestKeeper statistical algorithms. The results showed that ACT7, UBC and 18SrRNA were the stable reference genes in all of the tested samples. ACT7 and UBC showed high stability in different sampling times after the tribenuron treatment. UBC and 18SrRNA were the most suitable genes for different organs and growth stages. This work confirmed the suitable reference genes of flixweed for a relatively accurate gene expression analysis under different experimental conditions.

  1. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

    Directory of Open Access Journals (Sweden)

    Nour Hammoudeh

    2014-12-01

    Full Text Available Leishmania Homologue of receptors for Activated C Kinase (LACK antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica.The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR technique.The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed.Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.

  2. Preliminary Studies on Differential Expression of Auditory Functional Genes in the Brain After Repeated Blast Exposures

    Science.gov (United States)

    2012-01-01

    Army Medical Research and Materiel Command, Fort Detrick, MD Abstract—The mechanisms of central auditory processing involved in auditory/ vestibular ...trans- ducers in auditory neurons [22–23,45–48]. The frontal cor- tex and midbrain of blast-exposed mice showed significant increase in the expression of...auditory neurons [26]. Other types of molecules involved in calcium regula- tion, such as calreticulin and calmodulin-dependent pro- tein kinase expression

  3. Gene expression profiles in irradiated cancer cells

    Science.gov (United States)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-01

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  4. Gene expression profiles in irradiated cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C. [IBFM CNR - LATO, Cefalù, Segrate (Italy)

    2013-07-26

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  5. Gene expression analysis identifies global gene dosage sensitivity in cancer

    DEFF Research Database (Denmark)

    Fehrmann, Rudolf S. N.; Karjalainen, Juha M.; Krajewska, Malgorzata;

    2015-01-01

    expression. We reanalyzed 77,840 expression profiles and observed a limited set of 'transcriptional components' that describe well-known biology, explain the vast majority of variation in gene expression and enable us to predict the biological function of genes. On correcting expression profiles...... for these components, we observed that the residual expression levels (in 'functional genomic mRNA' profiling) correlated strongly with copy number. DNA copy number correlated positively with expression levels for 99% of all abundantly expressed human genes, indicating global gene dosage sensitivity. By applying...

  6. Noise in eukaryotic gene expression

    Science.gov (United States)

    Blake, William J.; KÆrn, Mads; Cantor, Charles R.; Collins, J. J.

    2003-04-01

    Transcription in eukaryotic cells has been described as quantal, with pulses of messenger RNA produced in a probabilistic manner. This description reflects the inherently stochastic nature of gene expression, known to be a major factor in the heterogeneous response of individual cells within a clonal population to an inducing stimulus. Here we show in Saccharomyces cerevisiae that stochasticity (noise) arising from transcription contributes significantly to the level of heterogeneity within a eukaryotic clonal population, in contrast to observations in prokaryotes, and that such noise can be modulated at the translational level. We use a stochastic model of transcription initiation specific to eukaryotes to show that pulsatile mRNA production, through reinitiation, is crucial for the dependence of noise on transcriptional efficiency, highlighting a key difference between eukaryotic and prokaryotic sources of noise. Furthermore, we explore the propagation of noise in a gene cascade network and demonstrate experimentally that increased noise in the transcription of a regulatory protein leads to increased cell-cell variability in the target gene output, resulting in prolonged bistable expression states. This result has implications for the role of noise in phenotypic variation and cellular differentiation.

  7. Identification of four soybean reference genes for gene expression normalization

    Science.gov (United States)

    Gene expression analysis requires the use of reference genes stably expressed independently of specific tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin and elongation factor 1-alpha) are commonly used as reference genes with the assumption tha...

  8. Evidence of inflammatory immune signaling in chronic fatigue syndrome: A pilot study of gene expression in peripheral blood

    Directory of Open Access Journals (Sweden)

    Vernon Suzanne D

    2008-09-01

    Full Text Available Abstract Background Genomic profiling of peripheral blood reveals altered immunity in chronic fatigue syndrome (CFS however interpretation remains challenging without immune demographic context. The object of this work is to identify modulation of specific immune functional components and restructuring of co-expression networks characteristic of CFS using the quantitative genomics of peripheral blood. Methods Gene sets were constructed a priori for CD4+ T cells, CD8+ T cells, CD19+ B cells, CD14+ monocytes and CD16+ neutrophils from published data. A group of 111 women were classified using empiric case definition (U.S. Centers for Disease Control and Prevention and unsupervised latent cluster analysis (LCA. Microarray profiles of peripheral blood were analyzed for expression of leukocyte-specific gene sets and characteristic changes in co-expression identified from topological evaluation of linear correlation networks. Results Median expression for a set of 6 genes preferentially up-regulated in CD19+ B cells was significantly lower in CFS (p = 0.01 due mainly to PTPRK and TSPAN3 expression. Although no other gene set was differentially expressed at p Conclusion Dissection of blood microarray profiles points to B cell dysfunction with coordinated immune activation supporting persistent inflammation and antibody-mediated NK cell modulation of T cell activity. This has clinical implications as the CD19+ genes identified could provide robust and biologically meaningful basis for the early detection and unambiguous phenotyping of CFS.

  9. Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus

    Directory of Open Access Journals (Sweden)

    Friedt Wolfgang

    2009-07-01

    Full Text Available Abstract Background Serial analysis of gene expression (LongSAGE was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus. The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana. Results Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP. Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development. Conclusion This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species.

  10. [Imprinting genes and it's expression in Arabidopsis].

    Science.gov (United States)

    Zhang, Hong-Yu; Xu, Pei-Zhou; Yang, Hua; Wu, Xian-Jun

    2010-07-01

    Genomic imprinting refers to the phenomenon that the expression of a gene copy depends on its parent of origin. The Arabidopsis imprinted FIS (Fertilisation-independent seed) genes, mea, fis2, and fie, play essential roles in the repression of central cell and the regulation of early endosperm development. fis mutants display two phenotypes: autonomous diploid endosperm development when fertilization is absent and un-cellularised endosperm formation when fertilization occurs. The FIS Polycomb protein complex including the above three FIS proteins catalyzes histone H3 K27 tri-methylation on target loci. DME (DEMETER), a DNA glycosylase, and AtMET1 (Methyltransferase1), a DNA methyltransferase, are involved in the regulation of imprinted expression of both mea and fis2. This review summarizes the studies on the Arabidopsis imprinted FIS genes and other related genes. Recent works have shown that the insertion of transposons may affect nearby gene expression, which may be the main driving force behind the evolution of genomic imprinting. This summary covers the achievements on Arabidopsis imprinted genes will provide important information for studies on genomic imprinting in the important crops such as rice and maize.

  11. Studies on the Cloning and Expression of Bbchit1 Gene of Beauveria bassiana NCIM 1216.

    Science.gov (United States)

    Pinnamaneni, Rajasekhar; Kalidas, P; Sambasiva Rao, K R S

    2011-07-01

    Beauveria bassiana is a biocontrol agent which shows entomopathogenecity on insect pests especially the Lepidopterons invading the agriculturally important crops. The mode of infection is through the cuticle by degrading the chitin present on it which is enabled by the exochitinase enzyme of Bbchit1 gene. A good quality genomic DNA was isolated from Beauveria bassiana NCIM 1216 and amplified with specific primers to isolate the gene corresponding to Bbchit1 which codes for the exochitinase enzyme that is responsible for pathogenesis. The Bbchit1 gene of B. bassiana was transformed with the binary plasmid pBANF-bar-pAN-Bbchit1, in which the Bbchit1 gene was placed downstream of the constitutive gpd promoter, which was mediated by A. tumefaciens, and transformants were selected on the basis of herbicide resistance. Fifty herbicide resistant colonies were obtained and analyzed. The exochitinase produced by these transformants was observed maximum on the 7th day of inoculation in both which was 0.09 μmol/ml/min for the purified fraction and 0.06 μmol/ml/min for the crude extract. The chitinolytic activity was observed maximum at pH 5 and at temperature of 40°C. The genetically modified pure form can be used in the production of transgenic plants and in bringing out commercial formulation for the control of Lepidopteran pests.

  12. Spatiotemporal patterns of gene expression during fetal monkey brain development.

    Science.gov (United States)

    Redmond, D Eugene; Zhao, Ji-Liang; Randall, Jeffry D; Eklund, Aron C; Eusebi, Leonard O V; Roth, Robert H; Gullans, Steven R; Jensen, Roderick V

    2003-12-19

    Human DNA microarrays are used to study the spatiotemporal patterns of gene expression during the course of fetal monkey brain development. The 444 most dynamically expressed genes in four major brain areas are reported at five different fetal ages. The spatiotemporal profiles of gene expression show both regional specificity as well as waves of gene expression across the developing brain. These patterns of expression are used to identify statistically significant clusters of co-regulated genes. This study demonstrates for the first time in the primate the relevance, timing, and spatial locations of expression for many developmental genes identified in other animals and provides clues to the functions of many unknowns. Two different microarray platforms are used to provide high-throughput cross validation of the most important gene expression changes. These results may lead to new understanding of brain development and new strategies for treating and repairing disorders of brain function.

  13. Genes Expressed in Human Tumor Endothelium

    Science.gov (United States)

    St. Croix, Brad; Rago, Carlo; Velculescu, Victor; Traverso, Giovanni; Romans, Katharine E.; Montgomery, Elizabeth; Lal, Anita; Riggins, Gregory J.; Lengauer, Christoph; Vogelstein, Bert; Kinzler, Kenneth W.

    2000-08-01

    To gain a molecular understanding of tumor angiogenesis, we compared gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues. Of over 170 transcripts predominantly expressed in the endothelium, 79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of these genes encode extracellular matrix proteins, but most are of unknown function. Most of these tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of anti-angiogenic therapies.

  14. Gene expression analysis of flax seed development

    Directory of Open Access Journals (Sweden)

    Sharpe Andrew

    2011-04-01

    clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.

  15. [Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. II. Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes].

    Science.gov (United States)

    Darbinian, N S; Popov, Iu G; Mochul'skiĭ, A V; Oming, D; Piruzian, E S; Vasilevko, V T

    1996-02-01

    The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum. The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively. These constructions were used for transformation of agrobacteria followed by transfer into plants. In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants. The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.

  16. Multi-organ expression profiling uncovers a gene module in coronary artery disease involving transendothelial migration of leukocytes and LIM domain binding 2: The Stockholm Atherosclerosis Gene Expression (STAGE) study

    KAUST Repository

    Hägg, Sara

    2009-12-04

    Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n =66/tissue) and atherosclerotic and unaffected arterial wall (n =40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n =15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n= 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n =49/48) and one visceral fat (n =59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P=0.008 and P=0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n =55/54) relating to carotid stenosis (P =0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n= 16/17, P<10 -27and-30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the Amodule was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the

  17. Multi-organ expression profiling uncovers a gene module in coronary artery disease involving transendothelial migration of leukocytes and LIM domain binding 2: the Stockholm Atherosclerosis Gene Expression (STAGE study.

    Directory of Open Access Journals (Sweden)

    Sara Hägg

    2009-12-01

    Full Text Available Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD. The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE study was to determine whether there are functionally associated genes (rather than individual genes important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue and atherosclerotic and unaffected arterial wall (n = 40/tissue isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes. In the second step (performed within tissue clusters, one atherosclerotic lesion (n = 49/48 and one visceral fat (n = 59 cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015. The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54 relating to carotid stenosis (P = 0.04, 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10(-27 and-30. Genes in the transendothelial migration of leukocytes (TEML pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module. In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004. The transcription co-factor LIM domain binding 2 (LDB2 was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2

  18. Expression profiles for six zebrafish genes during gonadal sex differentiation

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Morthorst, Jane E.; Andersen, Ole;

    2008-01-01

    the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. RESULTS...... the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three "male" genes (ar, sox9a and dmrt1) in the investigated period and 81% were high or low expressers of both...

  19. Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae

    Science.gov (United States)

    Vieira, Ana; Cabral, Ana; Fino, Joana; Azinheira, Helena G.; Loureiro, Andreia; Talhinhas, Pedro; Pires, Ana Sofia; Varzea, Vitor; Moncada, Pilar; Oliveira, Helena; Silva, Maria do Céu; Paulo, Octávio S.; Batista, Dora

    2016-01-01

    Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica—C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica—C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern

  20. Method Enabling Gene Expression Studies of Pathogens in a Complex Food Matrix

    DEFF Research Database (Denmark)

    Kjeldgaard, Jette; Henriksen, Sidsel; Cohn, Marianne Thorup

    2011-01-01

    We describe a simple method for stabilizing and extracting high-quality prokaryotic RNA from meat. Heat and salt stress of Escherichia coli and Salmonella spp. in minced meat reproducibly induced dnaK and otsB expression, respectively, as observed by quantitative reverse transcription-PCR (>5-fold...

  1. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been con...

  2. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Sternberg, Claus; Poulsen, Lars K.

    1998-01-01

    Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria ia is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been...

  3. Genes of periodontopathogens expressed during human disease.

    Science.gov (United States)

    Song, Yo-Han; Kozarov, Emil V; Walters, Sheila M; Cao, Sam Linsen; Handfield, Martin; Hillman, Jeffrey D; Progulske-Fox, Ann

    2002-12-01

    Since many bacterial genes are environmentally regulated, the screening for virulence-associated factors using classical genetic and molecular biology approaches can be biased under laboratory growth conditions of a given pathogen, because the required conditions for expression of many virulence factors may not occur during in vitro growth. Thus, technologies have been developed during the past several years to identify genes that are expressed during disease using animal models of human disease. However, animal models are not always truly representative of human disease, and with many pathogens, there is no appropriate animal model. A new technology, in vivo-induced antigen technology (IVIAT) was thus engineered and tested in our laboratory to screen for genes of pathogenic organisms induced specifically in humans, without the use of animal or artificial models of infection. This technology uses pooled sera from patients to probe for genes expressed exclusively in vivo (or ivi, in vivo-induced genes). IVIAT was originally designed for the study of Actinobacillus actinomycetemcomitans pathogenesis, but we have now extended it to other oral pathogens including Porphyromonas gingivalis. One hundred seventy-one thousand (171,000) clones from P. gingivalis strain W83 were screened and 144 were confirmed positive. Over 300,000 A. actinomycetemcomitans clones were probed, and 116 were confirmed positive using a quantitative blot assay. MAT has proven useful in identifying previously unknown in vivo-induced genes that are likely involved in virulence and are thus excellent candidates for use in diagnostic : and therapeutic strategies, including vaccine design.

  4. Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution

    Science.gov (United States)

    Erickson, Keesha E.; Otoupal, Peter B.

    2017-01-01

    ABSTRACT Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment

  5. Gene Expression Profiles for Predicting Metastasis in Breast Cancer: A Cross-Study Comparison of Classification Methods

    Directory of Open Access Journals (Sweden)

    Mark Burton

    2012-01-01

    Full Text Available Machine learning has increasingly been used with microarray gene expression data and for the development of classifiers using a variety of methods. However, method comparisons in cross-study datasets are very scarce. This study compares the performance of seven classification methods and the effect of voting for predicting metastasis outcome in breast cancer patients, in three situations: within the same dataset or across datasets on similar or dissimilar microarray platforms. Combining classification results from seven classifiers into one voting decision performed significantly better during internal validation as well as external validation in similar microarray platforms than the underlying classification methods. When validating between different microarray platforms, random forest, another voting-based method, proved to be the best performing method. We conclude that voting based classifiers provided an advantage with respect to classifying metastasis outcome in breast cancer patients.

  6. VEGF Gene Expression in Adult Human Thymus Fat: A Correlative Study with Hypoxic Induced Factor and Cyclooxigenase-2

    Science.gov (United States)

    Tinahones, Francisco; Salas, Julian; Mayas, María Dolores; Ruiz-Villalba, Adrian; Macias-Gonzalez, Manuel; Garrido-Sanchez, Lourdes; DeMora, Manuel; Moreno-Santos, Inmaculada; Bernal, Rosa; Cardona, Fernando; Bekay, Rajaa El

    2009-01-01

    It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT) produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties. Design Adult thymus fat and subcutaneous adipose tissue specimens were obtained from 28 patients undergoing cardiac surgery, making this tissue readily available as a prime source of adipose tissue. We focused our investigation on determining VEGF gene expression and characterizing the different genes, mediators of inflammation and adipogenesis, and which are known to play a relevant role in angiogenesis regulation. Results We found that VEGF-A was the isoform most expressed in TAT. This expression was accompanied by an upregulation of HIF-1α, COX-2 and HO-1 proteins, and by increased HIF-1 DNA binding activity, compared to SAT. Furthermore, we observed that TAT contains a high percentage of mature adipocytes, 0.25% of macrophage cells, 15% of endothelial cells and a very low percentage of thymocyte cells, suggesting the cellular variability of TAT, which could explain the differences in gene expression observed in TAT. Subsequently, we showed that the expression of genes known as adipogenic mediators, including PPARγ1/γ2, FABP-4 and adiponectin was similar in both TAT and SAT. Moreover the expression of these latter genes presented a significantly positive correlation with VEGF, suggesting the potential association between VEGF and the generation of adipose tissue in adult thymus. Conclusion Here we suggest that this fat has a potential angiogenic function related to ongoing adipogenesis, which substitutes immune functions within the adult thymus. The expression of VEGF seems to be associated with COX-2, HO-1 and adipogenesis related genes, suggesting the importance that this new fat has acquired in research in relation to

  7. VEGF gene expression in adult human thymus fat: a correlative study with hypoxic induced factor and cyclooxygenase-2.

    Directory of Open Access Journals (Sweden)

    Francisco Tinahones

    Full Text Available UNLABELLED: It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties. DESIGN: Adult thymus fat and subcutaneous adipose tissue specimens were obtained from 28 patients undergoing cardiac surgery, making this tissue readily available as a prime source of adipose tissue. We focused our investigation on determining VEGF gene expression and characterizing the different genes, mediators of inflammation and adipogenesis, and which are known to play a relevant role in angiogenesis regulation. RESULTS: We found that VEGF-A was the isoform most expressed in TAT. This expression was accompanied by an upregulation of HIF-1alpha, COX-2 and HO-1 proteins, and by increased HIF-1 DNA binding activity, compared to SAT. Furthermore, we observed that TAT contains a high percentage of mature adipocytes, 0.25% of macrophage cells, 15% of endothelial cells and a very low percentage of thymocyte cells, suggesting the cellular variability of TAT, which could explain the differences in gene expression observed in TAT. Subsequently, we showed that the expression of genes known as adipogenic mediators, including PPARgamma1/gamma2, FABP-4 and adiponectin was similar in both TAT and SAT. Moreover the expression of these latter genes presented a significantly positive correlation with VEGF, suggesting the potential association between VEGF and the generation of adipose tissue in adult thymus. CONCLUSION: Here we suggest that this fat has a potential angiogenic function related to ongoing adipogenesis, which substitutes immune functions within the adult thymus. The expression of VEGF seems to be associated with COX-2, HO-1 and adipogenesis related genes, suggesting the importance that this new

  8. Using RNA-Seq data to select refence genes for normalizing gene expression in apple roots

    Science.gov (United States)

    Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for t...

  9. Correlating Expression Data with Gene Function Using Gene Ontology

    Institute of Scientific and Technical Information of China (English)

    LIU,Qi; DENG,Yong; WANG,Chuan; SHI,Tie-Liu; LI,Yi-Xue

    2006-01-01

    Clustering is perhaps one of the most widely used tools for microarray data analysis. Proposed roles for genes of unknown function are inferred from clusters of genes similarity expressed across many biological conditions.However, whether function annotation by similarity metrics is reliable or not and to what extent the similarity in gene expression patterns is useful for annotation of gene functions, has not been evaluated. This paper made a comprehensive research on the correlation between the similarity of expression data and of gene functions using Gene Ontology. It has been found that although the similarity in expression patterns and the similarity in gene functions are significantly dependent on each other, this association is rather weak. In addition, among the three categories of Gene Ontology, the similarity of expression data is more useful for cellular component annotation than for biological process and molecular function. The results presented are interesting for the gene functions prediction research area.

  10. A transcriptomic study reveals differentially expressed genes and pathways respond to simulated acid rain in Arabidopsis thaliana.

    Science.gov (United States)

    Liu, Ting-Wu; Niu, Li; Fu, Bin; Chen, Juan; Wu, Fei-Hua; Chen, Juan; Wang, Wen-Hua; Hu, Wen-Jun; He, Jun-Xian; Zheng, Hai-Lei

    2013-01-01

    Acid rain, as a worldwide environmental issue, can cause serious damage to plants. In this study, we provided the first case study on the systematic responses of arabidopsis (Arabidopsis thaliana (L.) Heynh.) to simulated acid rain (SiAR) by transcriptome approach. Transcriptomic analysis revealed that the expression of a set of genes related to primary metabolisms, including nitrogen, sulfur, amino acid, photosynthesis, and reactive oxygen species metabolism, were altered under SiAR. In addition, transport and signal transduction related pathways, especially calcium-related signaling pathways, were found to play important roles in the response of arabidopsis to SiAR stress. Further, we compared our data set with previously published data sets on arabidopsis transcriptome subjected to various stresses, including wound, salt, light, heavy metal, karrikin, temperature, osmosis, etc. The results showed that many genes were overlapped in several stresses, suggesting that plant response to SiAR is a complex process, which may require the participation of multiple defense-signaling pathways. The results of this study will help us gain further insights into the response mechanisms of plants to acid rain stress.

  11. Pilot study on molecular quantitation and sequencing of endometrial cytokines gene expression and their effect on the outcome of in vitro fertilization (IVF cycle

    Directory of Open Access Journals (Sweden)

    D. Sabry

    2014-09-01

    Full Text Available Human trophoblast invasion and differentiation are essential for successful pregnancy outcome. The molecular mechanisms, however, are poorly understood. Interleukin (IL-11, a cytokine, regulates endometrial epithelial cell adhesion. Leukemia inhibitory factor (LIF is one of the key cytokines in the embryo implantation regulation. The present study aimed to assess the levels of LIF, IL-11, and IL-11 α receptor gene expression in the endometrium of women undergoing IVF and correlate their levels with the IVF pregnancy outcome. Also, the study aimed to detect any mutation in these three genes among IVF pregnant and non-pregnant women versus control menstrual blood of fertile women. Endometrial tissue biopsies were taken from 15 women undergoing IVF on the day of oocyte retrieval. The quantitative expression of IL-11, IL-11Rα, and LIF genes was assessed by real-time PCR and PCR products were sequenced. Menstrual blood from 10 fertile women was used as control to compare the DNA sequence versus DNA sequence of the studied genes in endometrial biopsies. LH, FSH, and E2 were assessed for enrolled patients by ELISA. Endometrial thickness was also assessed by pelvic ultrasonography. No significant difference was detected between quantitative expression of the three studied genes and pregnancy IVF outcome. Although DNA sequence changes were found in IL-11 and LIF genes of women with negative pregnancy IVF outcome compared to women with positive pregnancy IVF outcome, no DNA sequence changes were detected for IL-11Rα. Other studied parameters (e.g., age, LH, FSH, E2, and endometrial thickness showed no significant differences or correlation of quantitative expression of the three studied involved genes. Data suggested that there were no significant differences between quantitative expression of IL-11, IL-11Rα, and LIF genes and the IVF pregnancy outcome. The present study may reveal that changes in IL-11 and LIF genes sequence may contribute in

  12. Induction of immune gene expression and inflammatory mediator release by commonly used surgical suture materials: an experimental in vitro study.

    Science.gov (United States)

    Lock, Alistair M; Gao, Ryan; Naot, Dorit; Coleman, Brendan; Cornish, Jillian; Musson, David S

    2017-01-01

    Surgeons have a range of materials to choose from to complete wound closure, yet surprisingly very little is still known about the body's immune response to the suture materials in current use. The growing literature of adverse suture material reactions provided the objective of this study, to use in vitro assays to quantify levels of inflammation produced by seven commonly used suture materials in surgical procedures. Human monocyte/macrophage THP-1 cells were exposed to suture materials for 1, 3 and 5 days. Gene expression and protein secretion of six inflammatory cytokines and two cell surface markers were assessed using qPCR and ELISA respectively, with LPS exposure providing a positive control. Furthermore, a IL-1β/IL-1RA marker ratio was assessed to determine the balance between pro-/anti-inflammatory expression. The findings from our in vitro study suggest that four commonly used suture materials cause upregulation of pro-inflammatory markers indicative of an early foreign body reaction, with no balance from anti-inflammatory markers. As prolonged early pro-inflammation is known to produce delayed wound healing responses, the knowledge produced from this study has potential to improve informed surgical decision making and patient safety. This work has the capability to reduce suture-related adverse immune reactions, and therefore positively affect patient outcomes.

  13. Gene expression study of monocytes/macrophages during early foreign body reaction and identification of potential precursors of myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Lindsay Mesure

    Full Text Available Foreign body reaction (FBR, initiated by adherence of macrophages to biomaterials, is associated with several complications. Searching for mechanisms potentially useful to overcome these complications, we have established the signaling role of monocytes/macrophages in the development of FBR and the presence of CD34(+ cells that potentially differentiate into myofibroblasts. Therefore, CD68(+ cells were in vitro activated with fibrinogen and also purified from the FBR after 3 days of implantation in rats. Gene expression profiles showed a switch from monocytes and macrophages attracted by fibrinogen to activated macrophages and eventually wound-healing macrophages. The immature FBR also contained a subpopulation of CD34(+ cells, which could be differentiated into myofibroblasts. This study showed that macrophages are the clear driving force of FBR, dependent on milieu, and myofibroblast deposition and differentiation.

  14. A comparative analysis of green fluorescent protein and -glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

    Indian Academy of Sciences (India)

    P Kavita; Pradeep Kumar Burma

    2008-09-01

    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, -glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

  15. Fluid Mechanics, Arterial Disease, and Gene Expression.

    Science.gov (United States)

    Tarbell, John M; Shi, Zhong-Dong; Dunn, Jessilyn; Jo, Hanjoong

    2014-01-01

    This review places modern research developments in vascular mechanobiology in the context of hemodynamic phenomena in the cardiovascular system and the discrete localization of vascular disease. The modern origins of this field are traced, beginning in the 1960s when associations between flow characteristics, particularly blood flow-induced wall shear stress, and the localization of atherosclerotic plaques were uncovered, and continuing to fluid shear stress effects on the vascular lining endothelial) cells (ECs), including their effects on EC morphology, biochemical production, and gene expression. The earliest single-gene studies and genome-wide analyses are considered. The final section moves from the ECs lining the vessel wall to the smooth muscle cells and fibroblasts within the wall that are fluid me chanically activated by interstitial flow that imposes shear stresses on their surfaces comparable with those of flowing blood on EC surfaces. Interstitial flow stimulates biochemical production and gene expression, much like blood flow on ECs.

  16. Use of quantitative real time PCR for a genome-wide study of AYWB phytoplasma gene expression in plant and insect hosts

    DEFF Research Database (Denmark)

    Makarova, Olga; MacLean, Allyson M.; Hogenhout, Saskia A.

    2011-01-01

    Phytoplasmas are obligate parasites of plants and insects and cause significant crop yield losses worldwide. A number of microarray gene expression studies have been performed to understand better the effects of phytoplasma infection on plant physiology. However, little effort has been made...... this technique for reliable gene expression quantification of phytoplasmas on a large scale. In our experimental setup, 242 genes of aster yellows phytoplasma strain witches' broom (AY-WB) were tested for differences in expression in plant and insect host environments, and were shown to be predominantly...... expressed in the plant or insect hosts. In silico operon prediction corroborated the experimental data. Our findings suggest that the delta delta Ct method can be used to study the physiology of this pathogen...

  17. Stimulation of gene expression and loss of anular architecture caused by experimental disc degeneration--an in vivo animal study.

    Science.gov (United States)

    Guehring, Thorsten; Omlor, Georg W; Lorenz, Helga; Bertram, Helge; Steck, Eric; Richter, Wiltrud; Carstens, Claus; Kroeber, Markus

    2005-11-15

    An external compression model was used to evaluate gene and protein expression in intervertebral discs with moderate disc degeneration. To determine messenger ribonucleic acid and protein expression levels of relevant disc components. An animal model of mechanically induced disc degeneration was developed and characterized histologically. However, little is known at the molecular level in moderate disc degeneration. There were 8 New Zealand white rabbits subjected to monosegmental posterior compression to induce moderate disc degeneration. Twelve animals served as controls or sham controls. Discs were analyzed using immunohistochemistry for collagen type 1 (COL1), COL2, aggrecan, and bone morphogenetic protein-2/4 (BMP-2/4). For gene analysis, conventional and quantitative polymerase chain reactions were used for COL1A2, COL2A1, aggrecan, BMP-2, biglycan, decorin, osteonectin, fibromodulin, fibronectin, matrix metalloproteinase-13 (MMP-13), and tissue inhibitor of MMP-1. Gene expression for nontreated, sham-treated, and compressed discs was quantified in relation to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Immunohistochemistry of compressed discs showed a loss of anular architecture, and a significant reduction of BMP-2/4 and COL2 positive cells. Gene expression analysis showed a significant up-regulation of COL1A2, osteonectin, decorin, fibronectin, tissue inhibitor of MMP-1, BMP-2, and MMP-13 in compressed discs. Experimental moderate disc degeneration is characterized by a loss of BMP-2/4 and COL2 positive cells, although gene expression of disc constituents, catabolic enzymes, and growth factors is stimulated to reestablish disc integrity.

  18. Identification of several soybean cytosolic glutamine synthetase transcripts highly or specifically expressed in nodules: expression studies using one of the corresponding genes in transgenic Lotus corniculatus.

    Science.gov (United States)

    Marsolier, M C; Debrosses, G; Hirel, B

    1995-01-01

    A DNA fragment containing sequences hybridizing to the 5' region of GS15, a gene encoding soybean cytosolic glutamine synthetase, was isolated from a soybean genomic library. Mapping and partial sequence analysis of the genomic clone revealed that it encodes a cytosolic GS gene, GS21, which is different from GS15. In parallel, a number of cDNA clones encoding cytosolic GS were isolated using the coding region of pGS20 as a probe (pGS20 is a cDNA clone which corresponds to a transcript of the GS15 gene). Two new full-length cDNAs designated pGS34 and pGS38 were isolated and sequenced. In the 5' non-coding region a strong homology was found between the two clones and the GS21 gene. However, none of these sequences were identical, which suggests that there are at least three members in this group of genes. In order to determine their relative levels of transcription, specific sequences from pGS34, pGS38 and GS21 were used in an RNAse protection assay. This experiment clearly showed that GS21 and the gene encoding pGS38 are specifically expressed in young or mature nodules, whereas the gene encoding pGS34 is highly transcribed in nodules and constitutively expressed at a lower level in other soybean organs. In order to further analyse the molecular mechanisms controlling GS21 transcription, different fragments of the promoter region were fused to the Escherichia coli reporter gene encoding beta-glucuronidase (GUS) and the constructs were introduced into Lotus corniculatus via Agrobacterium rhizogenes-mediated transformation. Analysis of GUS activity showed that the GS21 promoter-GUS constructs were expressed in the vasculature of all vegetative organs. This result is discussed in relation to species-specific metabolic and developmental characteristics of soybean and Lotus.

  19. Analysis of multiplex gene expression maps obtained by voxelation

    Directory of Open Access Journals (Sweden)

    Smith Desmond J

    2009-04-01

    Full Text Available Abstract Background Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. Results To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in

  20. Gene expression signatures predict outcome in non-muscle invasive bladder carcinoma - a multi-center validation study

    DEFF Research Database (Denmark)

    Andersen, Lars Dyrskjøt; Zieger, Karsten; Real, Francisco X.

    2007-01-01

    PURPOSE: Clinically useful molecular markers predicting the clinical course of patients diagnosed with non-muscle-invasive bladder cancer are needed to improve treatment outcome. Here, we validated four previously reported gene expression signatures for molecular diagnosis of disease stage and ca...

  1. Left Ventricular Gene Expression Profile of Healthy and Cardiovascular Compromised Rat Models Used in Air Pollution Studies

    Science.gov (United States)

    The link between pollutant exposure and cardiovascular disease (CVD) has prompted mechanistic research with animal models of CVD. We hypothesized that the cardiac gene expression patterns of healthy and genetically compromised, CVD-prone rat models, with or without metabolic impa...

  2. Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Anna Francina, E-mail: Francina.Jackson@hc-sc.gc.ca [Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa K1S 5B6 (Canada); Williams, Andrew, E-mail: Andrew.Williams@hc-sc.gc.ca [Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9 (Canada); Recio, Leslie, E-mail: lrecio@ils-inc.com [ILS, Inc., P.O. Box 13501, Research Triangle Park, NC 27709 (United States); Waters, Michael D., E-mail: mwaters@ils-inc.com [ILS, Inc., P.O. Box 13501, Research Triangle Park, NC 27709 (United States); Lambert, Iain B., E-mail: Iain.Lambert@carleton.ca [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa K1S 5B6 (Canada); Yauk, Carole L., E-mail: Carole.Yauk@hc-sc.gc.ca [Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9 (Canada)

    2014-01-01

    Furan is a chemical hepatocarcinogen in mice and rats. Its previously postulated cancer mode of action (MOA) is chronic cytotoxicity followed by sustained regenerative proliferation; however, its molecular basis is unknown. To this end, we conducted toxicogenomic analysis of B3C6F1 mouse livers following three week exposures to non-carcinogenic (0, 1, 2 mg/kg bw) or carcinogenic (4 and 8 mg/kg bw) doses of furan. We saw enrichment for pathways responsible for cytotoxicity: stress-activated protein kinase (SAPK) and death receptor (DR5 and TNF-alpha) signaling, and proliferation: extracellular signal-regulated kinases (ERKs) and TNF-alpha. We also noted the involvement of NF-kappaB and c-Jun in response to furan, which are genes that are known to be required for liver regeneration. Furan metabolism by CYP2E1 produces cis-2-butene-1,4-dial (BDA), which is required for ensuing cytotoxicity and oxidative stress. NRF2 is a master regulator of gene expression during oxidative stress and we suggest that chronic NFR2 activity and chronic inflammation may represent critical transition events between the adaptive (regeneration) and adverse (cancer) outcomes. Another objective of this study was to demonstrate the applicability of toxicogenomics data in quantitative risk assessment. We modeled benchmark doses for our transcriptional data and previously published cancer data, and observed consistency between the two. Margin of exposure values for both transcriptional and cancer endpoints were also similar. In conclusion, using furan as a case study we have demonstrated the value of toxicogenomics data in elucidating dose-dependent MOA transitions and in quantitative risk assessment. - Highlights: • Global gene expression changes in furan-exposed mouse livers were analyzed. • A molecular mode of action for furan-induced hepatocarcinogenesis is proposed. • Key pathways include NRF2, SAPK, ERK and death receptor signaling. • Important roles for TNF-alpha, c-Jun, and NF

  3. Topological features in cancer gene expression data.

    Science.gov (United States)

    Lockwood, S; Krishnamoorthy, B

    2015-01-01

    We present a new method for exploring cancer gene expression data based on tools from algebraic topology. Our method selects a small relevant subset from tens of thousands of genes while simultaneously identifying nontrivial higher order topological features, i.e., holes, in the data. We first circumvent the problem of high dimensionality by dualizing the data, i.e., by studying genes as points in the sample space. Then we select a small subset of the genes as landmarks to construct topological structures that capture persistent, i.e., topologically significant, features of the data set in its first homology group. Furthermore, we demonstrate that many members of these loops have been implicated for cancer biogenesis in scientific literature. We illustrate our method on five different data sets belonging to brain, breast, leukemia, and ovarian cancers.

  4. Molecular Characteristics of High-Dose Melphalan Associated Oral Mucositis in Patients with Multiple Myeloma: A Gene Expression Study on Human Mucosa.

    Science.gov (United States)

    Marcussen, Mette; Bødker, Julie Støve; Christensen, Heidi Søgaard; Johansen, Preben; Nielsen, Søren; Christiansen, Ilse; Bergmann, Olav Jonas; Bøgsted, Martin; Dybkær, Karen; Vyberg, Mogens; Johnsen, Hans Erik

    2017-01-01

    Toxicity of the oral and gastrointestinal mucosa induced by high-dose melphalan is a clinical challenge with no documented prophylactic interventions or predictive tests. The aim of this study was to describe molecular changes in human oral mucosa and to identify biomarkers correlated with the grade of clinical mucositis. Ten patients with multiple myeloma (MM) were included. For each patient, we acquired three buccal biopsies, one before, one at 2 days, and one at 20 days after high-dose melphalan administration. We also acquired buccal biopsies from 10 healthy individuals that served as controls. We analyzed the biopsies for global gene expression and performed an immunohistochemical analysis to determine HLA-DRB5 expression. We evaluated associations between clinical mucositis and gene expression profiles. Compared to gene expression levels before and 20 days after therapy, at two days after melphalan treatment, we found gene regulation in the p53 and TNF pathways (MDM2, INPPD5, TIGAR), which favored anti-apoptotic defense, and upregulation of immunoregulatory genes (TREM2, LAMP3) in mucosal dendritic cells. This upregulation was independent of clinical mucositis. HLA-DRB1 and HLA-DRB5 (surface receptors on dendritic cells) were expressed at low levels in all patients with MM, in the subgroup of patients with ulcerative mucositis (UM), and in controls; in contrast, the subgroup with low-grade mucositis (NM) displayed 5-6 fold increases in HLA-DRB1 and HLA-DRB5 expression in the first two biopsies, independent of melphalan treatment. Moreover, different splice variants of HLA-DRB1 were expressed in the UM and NM subgroups. Our results revealed that, among patients with MM, immunoregulatory genes and genes involved in defense against apoptosis were affected immediately after melphalan administration, independent of the presence of clinical mucositis. Furthermore, our results suggested that the expression levels of HLA-DRB1 and HLA-DRB5 may serve as potential

  5. Evolution of Gene Expression Balance Among Homeologs of Natural Polyploids

    Directory of Open Access Journals (Sweden)

    Jasdeep S. Mutti

    2017-04-01

    Full Text Available Polyploidy is a major evolutionary process in eukaryotes, yet the expression balance of homeologs in natural polyploids is largely unknown. To study this expression balance, the expression patterns of 2180 structurally well-characterized genes of wheat were studied, of which 813 had the expected three copies and 375 had less than three. Copy numbers of the remaining 992 ranged from 4 to 14, including homeologs, orthologs, and paralogs. Of the genes with three structural copies corresponding to homeologs, 55% expressed from all three, 38% from two, and the remaining 7% expressed from only one of the three copies. Homeologs of 76–87% of the genes showed differential expression patterns in different tissues, thus have evolved different gene expression controls, possibly resulting in novel functions. Homeologs of 55% of the genes showed tissue-specific expression, with the largest percentage (14% in the anthers and the smallest (7% in the pistils. The highest number (1.72/3 of homeologs/gene expression was in the roots and the lowest (1.03/3 in the anthers. As the expression of homeologs changed with changes in structural copy number, about 30% of the genes showed dosage dependence. Chromosomal location also impacted expression pattern as a significantly higher proportion of genes in the proximal regions showed expression from all three copies compared to that present in the distal regions.

  6. Evolution of Gene Expression Balance Among Homeologs of Natural Polyploids.

    Science.gov (United States)

    Mutti, Jasdeep S; Bhullar, Ramanjot K; Gill, Kulvinder S

    2017-04-03

    Polyploidy is a major evolutionary process in eukaryotes, yet the expression balance of homeologs in natural polyploids is largely unknown. To study this expression balance, the expression patterns of 2180 structurally well-characterized genes of wheat were studied, of which 813 had the expected three copies and 375 had less than three. Copy numbers of the remaining 992 ranged from 4 to 14, including homeologs, orthologs, and paralogs. Of the genes with three structural copies corresponding to homeologs, 55% expressed from all three, 38% from two, and the remaining 7% expressed from only one of the three copies. Homeologs of 76-87% of the genes showed differential expression patterns in different tissues, thus have evolved different gene expression controls, possibly resulting in novel functions. Homeologs of 55% of the genes showed tissue-specific expression, with the largest percentage (14%) in the anthers and the smallest (7%) in the pistils. The highest number (1.72/3) of homeologs/gene expression was in the roots and the lowest (1.03/3) in the anthers. As the expression of homeologs changed with changes in structural copy number, about 30% of the genes showed dosage dependence. Chromosomal location also impacted expression pattern as a significantly higher proportion of genes in the proximal regions showed expression from all three copies compared to that present in the distal regions.

  7. Identification and validation of reference genes to study the gene expression in Gluconacetobacter diazotrophicus grown in different carbon sources using RT-qPCR.

    Science.gov (United States)

    Galisa, Péricles S; da Silva, Helder A P; Macedo, Aline V M; Reis, Verônica M; Vidal, Márcia S; Baldani, José I; Simões-Araújo, Jean L

    2012-10-01

    Gluconacetobacter diazotrophicus strain PAL5 is a nitrogen-fixing endophytic bacterium originally isolated from sugarcane and later on was found to colonize other plants such as rice, elephant grass, sweet potato, coffee, and pineapple. Currently, G. diazotrophicus has been considered a plant growth-promoting bacterium due to its characteristics of biological nitrogen fixation, phytohormone secretion, solubilization of mineral nutrients and antagonism to phytopathogens. Reverse transcription followed by quantitative real-time polymerase chain reaction (RT-qPCR) is a method applied for the quantification of nucleic acids because of its specificity and high sensitivity. However, the decision about the reference genes suitable for data validation is still a major issue, especially for nitrogen-fixing bacteria. To evaluate and identify suitable reference genes for gene expression normalization in the diazotrophic G. diazotrophicus, mRNA levels of fourteen candidate genes (rpoA, rpoC, recA, rpoD, fabD, gmk, recF, rho, ldhD, gyrB, gyrBC, dnaG, lpxC and 23SrRNA) and three target genes (matE, omp16 and sucA) were quantified by RT-qPCR after growing the bacteria in different carbon sources. The geNorm and Normfinder programs were used to calculate the expression stabilities. The analyses identified three genes, rho, 23SrRNA and rpoD, whose expressions were stable throughout the growth of strain PAL5 in the chosen carbon sources. In conclusion our results strongly suggest that these three genes are suitable to be used as reference genes for real-time RT-qPCR data normalization in G. diazotrophicus.

  8. Coevolution of gene expression among interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  9. Relations of adipose tissue CIDEA gene expression to basal metabolic rate, energy restriction, and obesity: population-based and dietary intervention studies.

    Science.gov (United States)

    Gummesson, Anders; Jernås, Margareta; Svensson, Per-Arne; Larsson, Ingrid; Glad, Camilla A M; Schéle, Erik; Gripeteg, Lena; Sjöholm, Kajsa; Lystig, Theodore C; Sjöström, Lars; Carlsson, Björn; Fagerberg, Björn; Carlsson, Lena M S

    2007-12-01

    Cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDEA) could be a potential target for the treatment of obesity via the modulation of metabolic rate, based on the findings that CIDEA inhibits the brown adipose tissue uncoupling process in rodents. Our objects were to investigate the putative link between CIDEA and basal metabolic rate in humans and to elucidate further the role of CIDEA in human obesity. We have explored CIDEA gene expression in adipose tissue in two different human studies: a cross-sectional and population-based study assessing body composition and metabolic rate (Mölndal Metabolic study, n = 92); and a longitudinal intervention study of obese subjects treated with a very low calorie diet (VLCD) (VLCD study, n = 24). The CIDEA gene was predominantly expressed in adipocytes as compared with other human tissues. CIDEA gene expression in adipose tissue was inversely associated with basal metabolic rate independently of body composition, age, and gender (P = 0.014). The VLCD induced an increase in adipose tissue CIDEA expression (P dysregulation of CIDEA expression was observed in individuals with the metabolic syndrome when compared with body mass index-matched controls. In a separate sample of VLCD-treated subjects (n = 10), uncoupling protein 1 expression was reduced during diet (P = 0.0026) and inversely associated with CIDEA expression (P = 0.0014). The findings are consistent with the concept that CIDEA plays a role in adipose tissue energy expenditure.

  10. Genome-Wide Association Study of Intelligence: Additive Effects of Novel Brain Expressed Genes

    Science.gov (United States)

    Loo, Sandra K.; Shtir, Corina; Doyle, Alysa E.; Mick, Eric; McGough, James J.; McCracken, James; Biederman, Joseph; Smalley, Susan L.; Cantor, Rita M.; Faraone, Stephen V.; Nelson, Stanley F.

    2012-01-01

    Objective: The purpose of the present study was to identify common genetic variants that are associated with human intelligence or general cognitive ability. Method: We performed a genome-wide association analysis with a dense set of 1 million single-nucleotide polymorphisms (SNPs) and quantitative intelligence scores within an ancestrally…

  11. Genome-Wide Association Study of Intelligence: Additive Effects of Novel Brain Expressed Genes

    Science.gov (United States)

    Loo, Sandra K.; Shtir, Corina; Doyle, Alysa E.; Mick, Eric; McGough, James J.; McCracken, James; Biederman, Joseph; Smalley, Susan L.; Cantor, Rita M.; Faraone, Stephen V.; Nelson, Stanley F.

    2012-01-01

    Objective: The purpose of the present study was to identify common genetic variants that are associated with human intelligence or general cognitive ability. Method: We performed a genome-wide association analysis with a dense set of 1 million single-nucleotide polymorphisms (SNPs) and quantitative intelligence scores within an ancestrally…

  12. Nasopharyngeal gene expression, a novel approach to study the course of respiratory syncytial virus infection

    NARCIS (Netherlands)

    van den Kieboom, Corné H; Ahout, Inge M L; Zomer, Aldert; Brand, Kim H; de Groot, Ronald; Ferwerda, Gerben; de Jonge, Marien I

    2014-01-01

    Respiratory syncytial virus (RSV) causes mild infections in the vast majority of children. However, in some cases, it causes severe disease, such as bronchiolitis and pneumonia. Development of severe RSV infection is determined by the host response. Therefore, the main aim of this study was to

  13. Differential Expression of Salinity Resistance Gene on Cotton

    Institute of Scientific and Technical Information of China (English)

    YE Wu-wei; YU Shu-xun

    2008-01-01

    @@ Salinity resistance and differential gene expression associated with salinity in cotton germplasm were studied,because of the large scale area of salinity in China,and its significant negative effects on the cotton production.The salinityresisted genes and their differential expression were studied under the stress of NaCI on cotton.There were found,under the NaCI stress,1644 genes differentially expressed from the salinity-sensitive cotton and only 817 genes differentially expressed from the salinityresisted cotton.

  14. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy (Davis, CA); Bachkirova, Elena (Davis, CA); Rey, Michael (Davis, CA)

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  15. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  16. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy [Davis, CA; Bachkirova, Elena [Davis, CA; Rey, Michael [Davis, CA

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  17. Methods for monitoring multiple gene expression